Use of Mutant Herpes Simplex Virus-2 for Cancer Therapy

ABSTRACT

The present invention is directed to the composition and use of a modified Herpes Simplex Virus Type 2 (HSV-2) as a medicament in the treatment of cancer. The modified HSV-2 has fusogenic activity, and comprises a modified/mutated ICP10 polynucleotide encoding a polypeptide having ribonucleotide reductase activity and lacking protein kinase activity.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to provisional application No.60/693,157 filed on Jun. 23, 2005, which is herein incorporated byreference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

The present invention was developed at least in part using fundsprovided by the United States Government pursuant to NIH grant numberRO1 CA 106671-01. The United States Government may have certain rightsin the invention.

FIELD OF THE INVENTION

The present invention is directed to the fields of virology, cancerbiology, cell biology, molecular biology, and medicine, including cancertherapeutics. Specifically, the present invention provides a mutantHerpes Simplex Virus-2 (HSV-2) comprising a modification of the ICP10gene and the use of this mutant HSV-2 for the treatment of malignantdiseases.

BACKGROUND OF THE INVENTION

Replication selective oncolytic viruses have shown great promise asanti-tumor agents for solid tumors. These viruses are able topreferentially replicate within tumor cells, while being restricted intheir ability to replicate in normal cells. The principle anti-tumormechanism of oncolytic viruses is through a direct cytopathic effect asthey propagate and spread from initially infected tumor cells tosurrounding tumor cells, achieving a larger volume of distribution andanticancer effects. Herpes simplex virus (HSV) has been modified foroncolytic purposes, most commonly by deleting viral genes necessary forefficient replication in normal (non-dividing) cells but not tumorcells. The modifications include deletion of either the viral γ34.5 geneor ICP6 gene. The viral γ34.5 gene functions as a neurovirulence factorduring HSV infection (Chou, et al, (1990) Science 250:1262-1266).Deletion of this gene blocks viral replication in non-dividing cells(McKie, et al., (1996) Br J Cancer 74(5): 745-52). The viral ICP6 geneencodes the large subunit of ribonucleotide reductase, which generatessufficient dNTP pools for efficient viral DNA replication and isabundantly expressed in tumor cells but not in non-dividing cells.Consequently, viruses with a mutation in this gene can preferentiallyreplicate in—and kill—tumor cells. The oncolytic HSV G207, which hasbeen extensively tested in animal studies and is currently in clinicaltrials, harbors deletions in both copies of the γ34.5 locus and aninsertional mutation in the ICP6 gene by the E. coli lacZ gene (Walker,et al., (1999) Human Gene Ther. 10(13):2237-2243). Alternatively, anoncolytic type-1 HSV can be constructed by using a tumor-specificpromoter to drive γ34.5 or other genes essential for HSV replication(Chung, et al., (1999) J Virol 73(9): 7556-64).

Oncolytic herpes simplex viruses (HSV) were initially designed andconstructed for the treatment of brain tumors (Andreansky, et al.,(1996) Proc Natl Acad. Sci. 93(21): 11313-11318). Subsequently, theyhave been found to be effective in a variety of other human solidtumors, including breast (Toda, et al., (1998) Human Gene Ther.9(15):2177-2185), prostate (Walker, et al., (1999) Human Gene Ther.10(13):2237-2243) lung (Toyoizumi, et al., (1999) Human Gene Ther.10(18):3013-3029), ovarian (Coukos, et al., (1999) Clin. Cancer Res.5(6):1523-1527), colon and liver cancers (Pawlik, et al., (2000) CancerRes. 61(11):2790-2795). The safety of oncolytic HSVs has also beenextensively tested in mice (Sundaresan, et al., (2000) J. Virol.74(8):3832-3841) and primates (Aotus), which are extremely sensitive toHSV infection (Todo, et al., (2000) Cancer Gene Ther. 7(6):939-946).These studies have confirmed that oncolytic HSVs are extremely safe forin vivo administration.

Oncolytic HSVs have been exclusively constructed from HSV-1. HSV-2 hasnot been explored for the purpose of constructing oncolytic viruses.Nonetheless, HSV-2 has some unique features that enhance its potentialas an oncolytic agent. For example, it has been reported that, unlikeHSV-1, HSV-2 encodes a secreted form of glycoprotein G (gG) that affectsthe function of neutrophils, monocyte and NK cells (Bellner, et al.(2005) J Immunol 174(4): 2235-41). Such a property may provide anoncolytic virus derived from HSV-2 with the ability to resist theinhibitory effect of the body's innate immunity. Innate immunity is aquick response of the host to invading microorganisms and it has beenfound to be the major factor that restricts HSV replication in vivo(Dalloul, et al., (2004) J Clin Virol 30(4): 329-36; Wakimoto, et al.,(2003) Gene Ther 10(11):983-90. Thus, an oncolytic virus derived fromHSV-2 should replicate and spread even when the patient's body developsanti-HSV innate immunity.

Despite encouraging preclinical studies, results from early clinicaltrials have suggested that the current versions of oncolytic viruses,although safe, may only have limited anti-tumor activity on their own(Nemunaitis, et al., (2001) J. Clin Oncol. 19(2):289-298). Studies fromthe inventors' work have demonstrated that incorporation ofcell-membrane fusion activity into an oncolytic HSV can dramaticallyimprove the anti-tumor potency of the virus (Fu, et al., (2002) Mol.Ther. 7(6): 748-754; Fu, et al., (2003) Cancer Res. 62: 2306-2312. Suchfusogenic oncolytic viruses produce syncytial formation in the tumor,directly enhancing the destructive power of the virus and promoting itsintra-tumor spread (Fu, et al., (2003) Cancer Res. 62: 2306-2312). Theuniquely combined tumor-destruction mechanism of syncytial formation anddirect cytolysis by the fusogenic oncolytic HSV also facilitates in situtumor antigen presentation, leading to potent anti-tumor immuneresponses (Nakamori, et al., (2004) Mol. Ther. 9(5): 658-665).Furthermore, the spread of a fusogenic oncolytic HSV through syncytialformation will allow it to maintain its anti-tumor activity even in thepresence of neutralizing anti-viral antibodies in the host. Viruses canonly replicate inside living cells and their replication usuallyrequires activation of certain cellular signaling pathways. Many viruseshave acquired various strategies during their evolution to activatethese signaling pathways to benefit their replication. The large subunitof herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP10or RR1) contains a unique amino-terminal domain which hasserine/threonine protein kinase (PK) activity. This PK activity has beenfound to activate the cellular Ras/MEK/MAPK pathway (Smith, et al.,(2000) J Virol 74(22): 10417-29).

Luo and Aurelian describe various vectors comprising different deletionsof the ICP10 gene in HSV-2 to demonstrate the relationship betweenparticular motifs and certain activities (Luo and Aurelian, (1992) JBiol Chem 267(14): 9645-53). Modified and deletion constructs of theHSV-2 ICP10 gene have been used to demonstrate particularcharacteristics of the ribonucleotide reductase domain (Peng et al.(1996) Virology 216(1): 184-96).

Deletion of the PK domain (ICP10 PK) from the ribonucleotide reductasegene severely compromises the ability of the virus to replicate in cellswhere there is no preexisting activated Ras signaling pathway (Smith etal (1998) J. Virol. 72(11):9131-9141).

U.S. Pat. No. 6,013,265 is directed to a vaccine that providesprotection from challenge by HSV-2, wherein the protein kinase domain ofICP10 has been deleted, which leads to deleterious effects on theability of HSV-2 to infect and transform cells.

The present invention fulfills a need in the art by providing noveltherapeutics for the treatment of cancer utilizing a modified HSV-2.

BRIEF SUMMARY OF THE INVENTION

The present invention addresses a long-felt need in the art by providinga potent modified Herpes Simplex Virus Type 2 (HSV-2), having oncolyticproperties. In specific embodiments of the invention, the virus has amodified ICP10 polynucleotide that encodes for an ICP10 polypeptide thathas ribonucleotide reductase activity, but lacks protein kinaseactivity. In particular aspects, the virus is useful for therapy ofmalignant cells. In specific embodiments, the virus replicatesselectively in tumor cells. In further specific embodiments, the virusgenerates cell membrane fusion to rid a culture, tissue, or organism ofat least some undesirable cells. In still further specific embodiments,the virus inhibits proliferation of at least some undesirable cells,and/or induces apoptosis in at least some desirable cells, and/orinduces a strong anti-tumor immune response, and/or a combinationthereof.

The native HSV-2 virus comprises an ICP10 polynucleotide (which may alsobe referred to as an RR1 polynucleotide) encoding a polypeptide havingan amino-terminal domain with protein kinase (PK) activity, such asserine/threonine protein kinase activity and a c-terminal domain havingribonucleotide reductase activity. In particular aspects of theinvention, the endogenous PK domain is modified such that the viruscomprises selective replication activity in tumor cells (and thereforecomprises activity to destroy tumor cells) and/or activity to render thevirus fusogenic or have enhanced fusogenic activity, in that itcomprises membrane fusion (syncytial formation) activity. In someembodiments, the ICP10 polynucleotide is modified by deleting at leastpart of the endogenous sequence encoding the protein kinase domain, suchthat the encoded polypeptide lacks protein kinase activity.

In another embodiment of the invention, a second polynucleotide replacesat least part of an endogenous ICP10 polynucleotide encoding for atleast part of the protein kinase domain. In yet other embodiments, theICP10 sequence that was not replaced comprises the entire RR domain. Thereplacement of at least part of the endogenous ICP10 polynucleotide mayoccur through any suitable method, such as for example, by homologousrecombination or other suitable genetic engineering methods, includingthe use of PCR and other methodologies as are well known to persons ofskill in the art.

In additional aspects of the invention, the polynucleotide that replacesat least part of the endogenous PK domain of ICP10 may be of anysuitable sequence. For example, the polynucleotide that replaces the PKdomain may encode a reporter gene product or a therapeutic gene product.The modified ICP10 polynucleotide containing the second polynucleotide(that replaced at least a portion of the PK domain) will encode for afusion protein comprised of the replacement polynucleotide and theremaining non-replaced portion of the ICP10 gene. Non-limiting examplesof reporter genes that are suitable for use with the present inventioninclude green fluorescent protein (SEQ ID NO:16; GenBank Accession No.U55761), β-galactosidase, luciferase, and Herpes simplex virus thymidinekinase (HSV-tk). Non-limiting examples of therapeutic polynucleotidesmay include Herpes simplex virus thymidine kinase (HSV-tk), cytosinedeaminase, caspase-3, and wild-type p53.

In still other embodiments of the invention, the polynucleotide thatreplaces at least part of the endogenous PK domain of ICP10 may be animmunomodulatory gene, or a polynucleotide that encodes for a fusogenicmembrane glycoprotein (FMG). Non-limiting examples of immunomodulatorygenes that are suitable for use with the present invention include IL-2,IL-12, or GM-CSF, and other cytokines; F42K and other cytokine analogs;or MIP-1, MIP-1 beta, MCP-1, RANTES, and other chemokines. Non-limitingexamples of polynucleotides encoding fusogenic membrane glycoproteinsthat are suitable for use with the present invention includeparamyxovirus F protein, HIV gp160 protein, SIV gp160 protein,retroviral Env protein, Ebola virus Gp, or the influenza virushaemagglutinin, a membrane glycoprotein from gibbon ape leukemia virus(GALV) or a C-terminally truncated form of the gibbon ape leukemia virusenvelope glycoprotein (GALV.fus).

In other embodiments of the invention, the modified ICP10 polynucleotideis operably linked to a constitutive promoter. Non-limiting exemplaryconstitutive promoters that are suitable for use with the presentinvention include the immediate early cytomegalovirus (CMV) promoter,SV40 early promoter, RSV LTR, Beta chicken actin promoter, and HSV0-TKpromoter. In other embodiments of the invention, the polynucleotide thatreplaces at least part of the endogenous PK domain (or TM domain)comprises a regulatory sequence operably linked thereto. The regulatorysequence is operable in a eukaryotic cell, in specific embodiments, andin further aspects is operable in a cancer cell. Non-limiting exemplarypromoters useful for practicing the methods and compositions describedherein may include tumor-specific promoters and/or tissue-specificpromoters, e.g., prostate-specific antigen (PSA) promoter, kallikrein 2promoter and probasin promoter (for prostate cancer), L-plastin promoter(for cancers of the breast, ovary and colon), thyroglobulin corepromoter (for thyroid carcinomas), Midkine and cyclooxygenase 2promoters (for pancreatic carcinoma), and human telomerase promoter(hTERT) for the majority of tumors.

In a further embodiment, there is provided a method of generating fusionbetween a first cell and a second cell, comprising the step of fusingthe second cell membrane with the first cell membrane by introducing tothe first cell a composition of the invention. In specific embodiments,the first cell, second cell, or both first and second cells aremalignant cells, such as those in a solid tumor. Non-limiting examplesof malignant cells suitable for use in practicing the methods andcompositions described herein may include breast cancer cells, lungcancer cells, skin cancer cells, prostate cancer cells, pancreaticcancer cells, colon cancer cells, brain cancer cells, liver cancercells, thyroid cancer cells, ovarian cancer cells, kidney cancer cells,spleen cancer cells, leukemia cells, or bone cancer cells.

In specific embodiments the introducing step is further defined asdelivering the virus to the human, such as by systemically deliveringthe virus to the human. Non-limiting routes of administration mayinclude administering the compositions described herein intravenously,intratumorally, intraperitoneally, or any combination thereof. Inspecific embodiments, the composition is introduced to a plurality ofcells.

In an additional embodiment, there is provided a method of destroying amalignant cell, such as one in a human, comprising the step ofintroducing to the cell a composition of the invention, whereinfollowing said introduction the membrane of the malignant cell fuseswith another cell membrane.

In another embodiment, there is a mammalian cell comprising acomposition of the invention. The mammalian cell may be a normallymphocyte, macrophage, natural killer cell or any other type of cellthat may function as a carrier to send the composition of the inventionto a tumor cell.

In yet another embodiment of the present invention, the modified HSV-2virus or viral vector as described herein induces apoptosis in cancercells infected with the virus. In yet another embodiment, apoptosis isinduced in bystander cells which are not infected with the virus, butsurround cells that are infected with the modified HSV-2 virus describedherein.

In yet another embodiment of the invention, a virus or viral vector asdescribed herein comprises part of a system for assaying the efficacy ofthe virus for lysing cells and or syncytial formation. The systemcomprises a cell contacted with a virus or vector as described herein.In some embodiments, the cell may be a eukaryotic cell, such as aprimary cancer cell, or a cell from a cancer cell line. In otherembodiments, the cell may be a prokaryotic cell that serves as host forthe virus or vector as described herein. In still other embodiments ofthe invention, the cell further comprising the virus or viral vector,may be maintained in vitro. In still other embodiments of the invention,the cell further comprising the virus or vector is placed into ananimal, such as a mouse. In still other embodiments of the invention,the cancer cell can be transplanted into an animal prior to being placedin contact with the virus or vector.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood. Itwill be appreciated by those skilled in the art that the conception andspecific embodiments disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It will also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features that are believed to be characteristic of theinvention, both as to its organization and method of operation, togetherwith further objects and advantages will be better understood from thefollowing description when considered in connection with theaccompanying examples and figures. It is to be expressly understood,however, that each of the examples and figures is provided for thepurpose of illustration and description only and is not intended as adefinition of the limits of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C shows the strategy for FusOn-H2 construction. FIG. 1A.Schematic representation of HSV-2 genome. The genome is represented by agray bar, while the terminal repeats (TR) and internal repeats (IR) areshown as gray boxes. The location of ICP10 gene is also indicated. FIG.1B. Enlarged view of the ICP10 gene, showing the positions of the PK andRR1 domains and the natural promoter. FIG. 1C. Modified ICP10 gene,which was subsequently inserted into the viral genome to constructFusOn-H2. As shown, the PK domain was replaced with the EGFP gene (inframe with the RR gene), and the original promoter of the gene wasreplaced with the immediate early promoter of cytomegalovirus, one ofthe strongest mammalian gene promoters. The BamHI restriction sites inthe unmodified and the modified ICP10 locus are labeled. The boxeslabeled as PKL, PK, GFP and PKR indicate the locations where the 4probes used in the Southern hybridization in FIG. 2 will hybridize to.

FIG. 2 shows a Southern blot analyses of FusOn-H2. Southern blothybridization, showing BamHI digested virion DNA from either theparental wild-type HSV-2 (w) or FusOn-H2 (m). The four probes used forthe Southern hybridization were: PKL, made from the left-flank; PK, madefrom the PK domain region; PKR, made from the right-flank region; GFP,prepared from the EGFP gene.

FIG. 3 shows a western blot analysis of FusOn-H2 using an anti-GFP mAb.Cell lysates were prepared from Vero cells infected with either FusOn-H2(m) or its parental wild-type HSV-2 (w), or from Vero cells transfectedwith pSZ-EGFP plasmid DNA (p).

FIG. 4 shows phenotypic characterization of FusOn-H2 in cultured cells.Cells were infected with the indicated viruses at 0.01 pfu/cell or leftuninfected. The micrographs were taken 24 h after infection. Thesyncytia are identified by white arrows. Among the cells tested,MDA-MB-435 is a human breast cancer line, MPans-96 is a human pancreaticcancer line and SKOV3 is a human ovarian cancer line. Originalmagnification: 200×.

FIGS. 5A-C shows selective replication of FusOn-H2. FIG. 5A. Vero cellswere maintained in fully cycling state (10% FBS) or were starved forserum for 24 h before they were infected with the viruses at 1 pfu/cell.Cells were harvested at the indicated time points and the virus yieldwas quantified by plaque assay on Vero cell monolayers. FIG. 5B. Verocells were incubated in medium containing a low percentage of serum (2%)alone or in the presence of 50 μM PD98059 during the virus infection.Cells were harvested at 24 h and 48 h after infection, and the foldreduction in virus replication was calculated by dividing the totalvirus yield in the well without PD98059 by that from the well containingthe drug. FIG. 5C. Primary hepatocytes cultured in vitro were infectedwith the indicated viruses at 1 pfu/cell. The viruses were harvested atthe indicated times after infection and quantified by plaque assay onVero cell monolayers. *p<0.01, FusOn-H2 compared with wt186 (Student'st-test).

FIGS. 6A and B. In vitro killing ability of human cancer cells byoncolytic HSVs. Cells were infected with the viruses at either 0.01pfu/cell (A) or 0.1 pfu/cell (B). Cell viability was determined with anLDH assay at the indicated times points. The percentage of cell killingwas calculated by dividing the LDH released from virus-infected cells bythat from uninfected cells; p<0.01, FusOn-H2 compared with wt186 orBaco-1; ^(Ψ)p<0.01, FusOn-H2 compared with wt186 (Student's t-test).

FIGS. 7A and B. In vivo anti-tumor activity of FusOn-H2 againstxenografted human breast cancer. FIG. 7A. Therapeutic effect afterintra-tumor delivery. Human breast tumor xenografts were established byinjecting MDA-MB-435 cells into the fat part of the second mammary. Whentumors reached about 5 mm in diameter, viruses were injectedintratumorally at a dose of 1×10⁶ pfu. Treatment groups includeFusOn-H2, Baco-1, or PBS. The tumor growth ratio was determined bydividing the tumor volume measured on the indicated week after virusinjection by the tumor volume before treatment (n=8 mice per group).FIG. 7B. Therapeutic effect against large breast tumor xenografts.Tumors were 10 and 10-15 mm in diameter, respectively, for intra-tumorand intravenous injection groups (n=5 each). For intra-tumor andintravenous injections, viruses were given at doses of 3×10⁶ pfu and1.5×10⁷ pfu, respectively. The tumor growth ratio was calculated in thesame way as in FIG. 6A. ^(Ψ)p<0.05, FusOn-H2 compared with Baco-1;*p<0.01, FusOn-H2 compared with Synco-2D (Student's t-test).

FIG. 8. Therapeutic effect of FusOn-H2 against metastatic human ovariancancer xenografts established in the peritoneal cavity of nude mice.Human ovarian cancer xenografts were established by intraperitonealinoculation of 2×10⁶ SKOV3 cells into the peritoneal cavity (n=8 miceper treatment group). Eight and 15 days after tumor cell inoculation,the mice received an intraperitoneal injection of oncolytic HSVs at adose of 3×10⁶ pfu, at a site distant from the tumor implantation site.Four weeks after the initial virus injection (i.e., 5 weeks after tumorcell implantation), the mice were euthanized. The gross appearance ofthe tumor nodules is shown in this figure while the number of tumornodules and the tumor weight from each animal are shown in Table 1.

DETAILED DESCRIPTION OF THE INVENTION

The HSV-2 viral composition as described in Example 1, was deposited onJun. 8, 2006, with the American Type Culture Collection (ATCC) 10801University Blvd. Manassas, Va. 20110-2209 USA. The ATCC is anInternational Depository Authority (IDA) as established under theBudapest Treaty. The certificate of deposit number is ______.

I. DEFINITIONS

The term “Herpes Simplex Virus” or “HSV” as used herein refers to anenveloped, icosahedral, double-stranded DNA virus that infects mammals,including humans. Wild-type HSV infects and replicates in bothterminally differentiated non-dividing cells and dividing cells. “HSV-2”refers to a member of the HSV family that contains the ICP10 gene. Theterm “FusOn-H2” as used herein refers to a HSV-2 mutant having amodified ICP10 polynucleotide encoding a polypeptide havingribonucleotide reductase activity, but lacking protein kinase activityas described herein.

The term “cell membrane fusion” as used herein refers to fusion of anouter membrane of at least two cells, such as two adjacent cells, forexample.

The term “enhanced fusogenic activity” as used herein refers to anenhancement, increase, intensification, argumentation, amplification, orcombination thereof of the cell membrane fusion.

The term “oncolytic” as used herein refers to a property of an agentthat can result directly or indirectly, in the destruction of malignantcells. In a specific embodiment, this property comprises causing fusionof a malignant cell membrane to another membrane.

The term “replication selective” or “replication conditional” as usedherein refers to the ability of an oncolytic virus to selectively growin certain tissues (e.g., tumors).

The term “syncytium” as used herein refers to a multinucleate giant cellformation involving a significantly larger number of fused cells.

The term “vector” as used herein refers to a carrier nucleic acidmolecule into which a nucleic acid sequence can be inserted forintroduction into a cell where it can be replicated. The insertednucleic acid sequence is referred to as “exogenous” either when it isforeign to the cell into which the vector is introduced or when it ishomologous to a sequence in the cell but in a position within the hostcell nucleic acid in which die sequence is ordinarily not found. Avector can be either a non-viral DNA vector or a viral vector. Viralvectors are encapsulated in viral proteins and capable of infectingcells. non-limiting examples of vectors include: a viral vector, anon-viral vector, a naked DNA expression vector, a plasmid, a cosmid, anartificial chromosome (e.g., YACS), a phage-vector, a DNA expressionvector associated with a cationic condensing agent, a DNA expressionvector encapsulated in a liposome, or a certain eukaryotic cell e.g., aproducer cell. Unless stated otherwise, “vector” as used herein refersboth a DNA vector and a viral vector. One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques. Generally, these include Sambrook et al., Molecular Cloning:A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989)and the references cited therein. Virological considerations are alsoreviewed in Coen D. M, Molecular Genetics of Animal Viruses in Virology,2^(nd) Edition, B. N. Fields (editor), Raven Press, N.Y. (1990) and thereferences cited therein.

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for a RNA capable of being transcribed.In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind, such as RNA polymerase and other transcriptionfactors to initiate or regulate the temporal and spatial transcriptionof a nucleic acid sequence. The phrases “operatively positioned,”“operably linked,” “under control,” and “under transcriptional control”mean that a promoter is in a correct functional location and/ororientation in relation to a nucleic acid sequence to controltranscriptional initiation and/or expression of that sequence. Exemplarynon-limiting promoters include: a constitutive promoter, atissue-specific promoter, a tumor-specific promoter, or an endogenouspromoter under the control of an exogenous inducible element.

The term “constitutive promoter” as used herein refers to a promoterthat drives expression of a gene or polynucleotide in a continuoustemporal manner throughout the cell cycle. A constitutive promoter maybe cell or tissue-type specific as long as it operates in a continuousfashion throughout the cell cycle to drive the expression of the gene orpolynucleotide with which it is associated. Exemplary non-limitingconstitutive promoters include: the immediate early cytomegalovirus(CMV) promoter, SV40 early promoter, RSV LTR, Beta chicken actinpromoter, and HSV TK promoter.

The term “enhancer” refers to a cis-acting regulatory sequence involvedin the control of transcriptional activation of a nucleic acid sequence.

The terms “contacted” and “exposed,” when applied to a cell are usedherein to describe the process by which a virus, viral vector, non-viralvector, DNA vector, or any other therapeutic agent, alone or incombination, is delivered to a target cell or placed in directjuxtaposition with a target cell.

The phrase “modified ICP10 polynucleotide” refers to an ICP10polynucleotide that encodes for an ICP10 polypeptide that hasribonucleotide reductase (RR) activity, but lacks protein kinaseactivity.

The phrase “ribonucleotide reductase activity” refers to ability of theC-terminal domain of the polypeptide encoded by an ICP10 polynucleotideto generate sufficient deoxynucleotide triphosphates (dNTPs) requiredfor viral replication.

The phrase “protein kinase activity” refers to the ability of theamino-terminal domain of the polypeptide encoded by an ICP10polynucleotide to phosphorylate serine and threonine residues capable ofactivating the Ras/MEK/MAPK pathway.

The term “by-stander tumor cell” as used herein refers to tumor cellsthat are not infected with a modified HSV-2 virus as described herein,but are adjacent to or near tumor cells that are infected with a virusor vector as described herein.

The term “anti-cancer agent” as used herein refers to an agent that iscapable of negatively affecting cancer in a subject, for example, bykilling cancer cells, inducing apoptosis in cancer cells, reducing thegrowth rate of cancer cells, reducing the incidence or number ofmetastases, reducing tumor size, inhibiting tumor growth, reducing theblood supply to a tumor or cancer cells, promoting an immune responseagainst cancer cells or a tumor, preventing or inhibiting theprogression of cancer, or increasing the lifespan of a subject withcancer.

The phrases “pharmaceutically” or “pharmacologically acceptable” as usedherein refer to molecular entities and compositions that do not producean adverse, allergic or other untoward reaction when administered to ananimal, or human, as appropriate. The phrase “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like.

The term “unit dose” refers to a physically discrete unit suitable foruse in a subject, each unit containing a predetermined quantity of thetherapeutic composition calculated to produce the desired response inassociation with its administration, i.e., the appropriate route andtreatment regimen.

The term “effective” or “therapeutically effective” as used hereinrefers to inhibiting an exacerbation in symptoms, preventing onset of adisease, preventing spread of disease, amelioration of at least onesymptom of disease, or a combination thereof.

II. INTRODUCTION

Viruses can only replicate inside living cells and their replicationusually requires activation of certain cellular signaling pathways. Manyviruses have acquired various strategies during their evolution toactivate these signaling pathways to benefit their replication. Thelarge subunit of herpes simplex virus type 2 (HSV-2) ribonucleotidereductase (ICP10 or RR1) comprises a unique amino-terminal domain thathas serine/threonine protein kinase (PK) activity. This PK activity hasbeen found to activate the cellular Ras/MEK/MAPK pathway (Smith, et al.,(2000) J Virol 74(22): 10417-29). Consequently, it has been reportedthat deletion of this PK domain (ICP10 PK) from the ribonucleotidereductase gene severely compromises the ability of the virus toreplicate in cells, such as those where there is no preexistingactivated Ras signaling pathway (Smith, et al., (1998) J. Virol.72(11):9131-9141).

Here, the present inventors show that when the PK domain of HSV-2 isreplaced and/or modified such that protein encoded by the modified ICP10gene has ribonucleotide reductase activity, but lacks protein kinaseactivity, the virus selectively replicates in and destroys tumor cells(at least tumor cells in which the Ras signaling pathway isconstitutively activated due to tumorigenesis). Furthermore,modification of the ICP10 polynucleotide as described herein renders thevirus intrinsically fusogenic, i.e., infection of tumor cells with thevirus induces widespread cell membrane fusion (syncytial formation).This property increases the destructive power of the virus against tumorcells. Furthermore, in vivo studies show that this virus is extremelysafe for either local or systemic administration.

In some embodiments of the invention, the modification of the PK domaincomprises insertion of a reporter gene, such as that expressing thegreen fluorescent gene, and/or replacement of the native promoter genewith a constitutive promoter, such as the immediate earlycytomegalovirus promoter.

In some embodiments, the HSV-2 is genetically engineered either byinserting a second polynucleotide into the polynucleotide encoding theprotein kinase activity domain of the ICP10 gene, or by replacing aportion of the protein kinase domain with a second polynucleotide suchthat the polypeptide encoded by the modified polynucleotide hasribonucleotide reductase activity, but lacks protein kinase activity.For example, the second polynucleotide may encode a glycoprotein, suchas a fusogenic membrane glycoprotein. A preferred glycoprotein for usewithin the scope of the present invention is a truncated form of gibbonape leukemia virus envelope fusogenic membrane glycoprotein (GALV.fus).In certain aspects of the invention, expression of GALV.fus in thecontext of the oncolytic virus of the present invention significantlyenhances the anti-tumor effect of the virus.

In some embodiments, the modified HSV-2 of the invention comprises amutation, such as a deletion, in ICP10 that provides cell fusogenicproperties to the virus. Such a mutation may be generated randomlyduring the virus screening or obtained from nature, and a pool ofpotential candidates for having cell fusogenic properties is thenassayed for the function by means described herein and/or known in theart. A mutation leading to the fusogenic phenotype may be a pointmutation, a frame shift, an inversion, a deletion, a splicing errormutation, a post-transcriptional processing mutation, over expression ofcertain viral glycoproteins, a combination thereof, and so forth. Themutation may be identified by sequencing the particular HSV-2 andcomparing it to a known wild type sequence.

The modified HSV-2 of the present invention is useful for the treatmentof malignant cells, such as, for example, to inhibit their spread,decrease or inhibit their division, eradicate them, prevent theirgeneration or proliferation, or a combination thereof. The malignantcells may be from any form of cancer, such as a solid tumor, althoughother forms are also treatable. The modified HSV-2 of the presentinvention is useful for the treatment of lung, liver, prostate, ovarian,breast, brain, pancreatic, testicular, colon, head and neck, melanoma,and other types of malignancies. The invention is useful for treatingmalignant cells at any stage of a cancer disease, including metastaticstages of the disease. The invention may be utilized as a stand-alonetherapy or in conjunction with another means of therapy, includingchemotherapy, surgery, radiation, and the like.

III. MODIFIED ICP10 POLYNUCLEOTIDE

The present invention describes a HSV-2 mutant having a modified ICP10polynucleotide, wherein the modified ICP10 polynucleotide encodes for apolypeptide that has ribonucleotide reductase activity, but lacksprotein kinase (PK) activity. The ICP10 polynucleotide may be modifiedeither by deleting at least some of the sequence required for encoding afunctional PK domain, or replacing at least part of the sequenceencoding the PK domain with a second polynucleotide. One of skill in theart will recognize that any suitable method can be used for generatingthe modified ICP10 polynucleotide, including mutagenesis, polymerasechain reaction, homologous recombination, or any other geneticengineering technique known to a person of skill in the art.

A. Mutagenesis

In specific embodiments of the invention, an ICP10 sequence of an HSV-2virus, is mutated, such as by deletion, using any of a variety ofstandard mutagenic procedures. Mutation can involve modification of anucleotide sequence, a single gene, or blocks of genes. A mutation mayinvolve a single nucleotide (such as a point mutation, which involvesthe removal, addition or substitution of a single nucleotide base withina DNA sequence) or it may involve the insertion or deletion of largenumbers of nucleotides. Mutations can arise spontaneously as a result ofevents such as errors in the fidelity of DNA replication, or inducedfollowing exposure to chemical or physical mutagens. A mutation can alsobe site-directed through the use of particular targeting methods thatare well known to persons of skill in the art.

B. Genetic Recombination

In other embodiments of the invention, the ICP10 polynucleotide ismodified using genetic recombination techniques to delete or replace atleast part of the sequence encoding for the PK domain. The region of thePK domain that is deleted/replaced may be any suitable region so long asthe polypeptide encoded by the modified ICP10 polynucleotide retainsribonucleotide reductase activity and lacks protein kinase activity. Incertain embodiments, though, the modification to the PK domain affectsone or more of the eight PK catalytic motifs (amino acid residues106-445, although the PK activity may be considered amino acid residues1-445), and/or the transmembrane (TM) region, and/or the invariant Lys(Lys 176). An exemplary wild-type ICP10 polypeptide sequence is providedin SEQ ID NO:15 (National Center for Biotechnology Information's GenBankdatabase Accession No. 1813262A). An exemplary wild-type polynucleotidethat encodes an ICP10 polypeptide is provided in SEQ ID NO:17.

In certain embodiments, the ICP10 polynucleotide is modified by merelydeleting a portion of the sequence encoding the PK domain that isnecessary for PK activity. An exemplary ICP10 polynucleotide lacking atleast some sequence that encodes a PK domain is provided in SEQ IDNO:18. In another exemplary embodiment, ICP10 polynucleotide is modifiedsuch that the PK domain is deleted in its entirety, as provided in SEQID NO:19. Both SEQ ID NO:18 and SEQ ID NO:19 are suitable for use ingenerating a HSV-2 mutant as described herein, as both sequences encodefor polypeptides that have ribonucleotide reductase activity, but lackprotein kinase activity. In certain embodiments of the invention, themodified ICP10 polynucleotide disclosed in SEQ ID NO:18 or SEQ ID NO:19may be under the control of the endogenous HSV-2 promoter, or operablylinked to a constitutive promoter, such as the immediate earlycytomegalovirus promoter described in SEQ ID NO:20.

In still other embodiments of the invention, the ICP10 polynucleotide ismodified by replacing at least part of the sequence encoding the PKdomain with a second polynucleotide, such as green fluorescent protein,which is placed in frame with the sequence encoding the RR domain of theICP10 polynucleotide. This construct can be either under control of theendogenous HSV-2 promoter, or under the control of a constitutivepromoter such as the CMV promoter (SEQ ID NO:20). This latter construct(containing GFP replacement polynucleotide and the CMV promoter) isdescribed in greater detail in Example 1.

In another aspect of the invention, the polynucleotide that replaces atleast part of the protein kinase activity domain of the endogenous ICP10in HSV-2 can encode at least a fusogenic portion of a cell membranefusion-inducing polypeptide, such as a viral fusogenic membraneglycoprotein (FMG). The polypeptide is preferably capable of inducingcell membrane fusion at a substantially neutral pH (such as about pH6-8), for example.

In particular embodiments, the FMG comprises at least a fusogenic domainfrom a C-type retrovirus envelope protein, such as MLV (as an example,SEQ ID NO:6) or GALV (as an example, SEQ ID NO:5). A retroviral envelopeprotein having a deletion of some, most, or all of the cytoplasmicdomain is useful, because such manipulation results in hyperfusogenicactivity for human cells. Particular modifications are introduced, insome embodiments, into viral membrane glycoproteins to enhance theirfunction to induce cell membrane fusion. For example, truncation of thecytoplasmic domains of a number of retroviral and herpes virusglycoproteins has been shown to increase their fusion activity,sometimes with a simultaneous reduction in the efficiency with whichthey are incorporated into virions (Rein et al., (1994) J Virol 68(3):1773-81).

Some examples of cell membrane fusing polypeptides include measles virusfusion protein (SEQ ID NO:7), the HIV gp160 (SEQ ID NO:8) and SIV gp160(SEQ ID NO:9) proteins, the retroviral Env protein (SEQ ID NO:10), theEbola virus Gp (SEQ ID NO:11), and the influenza virus haemagglutinin(SEQ ID NO:12).

In other embodiments, a second functional polynucleotide may be eitherinserted into the PK domain, or used to replace part or all of the PKdomain. This second functional polynucleotide may encode for animmunomodulatory or other therapeutic agent. It is contemplated thatthese additional agents will affect the upregulation of cell surfacereceptors and GAP junctions, cytostatic and differentiation agents,inhibit cell adhesion, or increase the sensitivity of the malignantcells to apoptosis. Exemplary, non-limiting examples of polynucleotidesencoding for immunomodulatory or other therapeutic agents include tumornecrosis factor; interferon, alpha, beta, gamma; interleukin-2 (IL-2),IL-12, granulocyte macrophage-colony stimulating factor (GM-CSF), F42K,MIP-1, MIP-1β, MCP-1, RANTES, Herpes Simplex Virus-thymidine kinase(HSV-tk), cytosine deaminase, and caspase-3.

In still other embodiments of the invention, the ICP10 polynucleotide ismodified by insertion of a polynucleotide encoding a reporter protein.Exemplary non-limiting polynucleotides encoding for reporter proteinsinclude green fluorescent protein, enhanced green fluorescent protein,β-galactosidase, luciferase, and HSV-tk.

C. Ribonucleotide Reductase Activity Assay

The biologic activity of RR can be detected as previously described(Averett, et al., J. Biol. Chem. 258:9831-9838 (1983) and Smith et al.,J. Virol. 72:9131-9141 (1998)) with the following modifications. BHKcells are initially grown to confluence in complete GMEM (containing 10%FBS) and then incubated for three days in 0.5% FBS EMEM, followed byinfection with 20 pfu of wild-type HSV, HSV-2 mutant, or mock infection.The cells are harvested 20 hours post infection, resuspended in 500 μlHD buffer [100 mM HEPES buffer (pH 7.6), 2 mM dithiothreitol (DTT)] andincubated on ice for 15 minutes before a 30 second sonication. Celldebris is cleared by centrifugation (16,000 g, 20 minutes, 4° C.) andthe supernatant is precipitated with crystalline ammonium sulfate at 45%saturation (0.258 g/ml). After a second centrifugation (16,000 g, 30minutes), the pellets are dissolved in 100 μl HD buffer, from which 50μl is taken to mix with an equal volume of 2× reaction buffer (400 mMHEPES buffer (pH 8.0), 20 mM DTT and 0.02 mM [³H]-CDP (24 Ci/mmol,Amersham, Chicago, Ill.). The reaction is terminated by the addition of100 mM hydroxyurea with 10 mM EDTA (pH 8.0) and boiling for 3 minutes.Then 1 ml of Crotalux atrox venom (Sigma, St. Louis, Mo.) is added andincubated for 30 minutes at 37° C., followed by another 3 minuteboiling. The solution is then passed through a 0.5 ml Dowex-1 boratecolumn, and samples eluted with 2 ml water and collected in four elutionfractions for scintillation counting after mixing with Biofluor (NewEngland Nuclear, Boston, Mass.). Ribonucleotide reductase activity isexpressed as units/mg protein where 1 unit represents the conversion of1 nmol [³H]CDP to dCDP/hr/mg protein.

D. Protein Kinase Activity Assay

To determine whether the modified ICP10 polynucleotide encodes apolypeptide that lacks protein kinase activity, extracts of cellsinfected with HSV-2 having a modified ICP10 polynucleotide or wild-typeHSV-2 (moi=200, 16 hours post infection) are immunopercipitated withanti LA-1 antibody and subjected to PK assays as described in Chung etal. J. Virol. 63:3389-3398, 1998 and U.S. Pat. No. 6,013,265. Generally,immunopercipitates of cell extracts are normalized for proteinconcentration using a BCA protein assay kit (PIERCE, Rockford Ill.)washed with TS buffer containing 20 mM Tris-HCL (pH 7.4), 0.15 M NaCl,suspended in 50 μl kinase reaction buffer consisting of 20 mM Tris-HCL(pH 7.4) 5 mM MgCl₂, 2 mM Mn Cl₂, 10 μCi [³²p] ATP (3000 Ci/mmol,DuPont, New England Research Prod.) and incubated at 30° C. for 15minutes. The beads are washed once with 1 ml TS buffer, resuspended in100 μl denaturing solution and boiled for 5 minutes. Proteins are thenresolved by SDS-PAGE on a 7% polyacrylamide gel. Proteins are thenelectrotransferred onto nitrocellulose membranes as previously described(see, Aurelian et. al., Cancer Cells 7:187-191 1989) and immunoblottedby incubation with specific antibodies followed by protein A-peroxidase(Sigma, St. Louis, Mo.) for 1 hour at room temperature. Detection can bemade with ECL reagents (Amersham, Chicago, Ill.) as described in Smithet al., Virol. 200:598-612, (1994).

IV. VECTOR CONSTRUCTION

The present invention is directed to an HSV-2 vector comprising areplacement or deletion of at least part of an ICP10 sequence, such thatthe protein encoded for by the modified ICP10 polynucleotide hasribonucleotide reductase activity, but lacks protein kinase activity,and in specific embodiments further comprising a regulatory sequence,such as a constitutive promoter. In some embodiments, the composition isa naked (non-viral) DNA vector comprising the modified ICP10 gene, andin other embodiments, the composition is a recombinant HSV-2 having themodified ICP10 gene. Both the naked DNA vector, and the recombinantvirus can be further comprised of some or all of the followingcomponents.

A. Vectors

Vectors, as defined supra, include but are not limited to plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). Methods for the construction ofengineered viruses and DNA vectors are known in the art. Generally theseinclude Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Press (1989) and the references citedtherein. Virological considerations are also reviewed in Coen D. M,Molecular Genetics of Animal Viruses in Virology, 2.sup.nd Edition, B.N. Fields (editor), Raven Press, N.Y. (1990) and the references citedtherein.

Expression vectors can contain a variety of “control sequences,” whichrefer to nucleic acid sequences necessary for the transcription andpossibly translation of an operably linked coding sequence in aparticular host cell. In addition to control sequences that governtranscription and translation, DNA vectors, expression vectors, andviruses may contain nucleic acid sequences that serve other functions aswell and are described infra.

1. Promoters and Enhancers

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. The best-known example of this is the TATAbox, but in some promoters lacking a TATA box (e.g., the promoter forthe mammalian terminal deoxynucleotidyl transferase gene and thepromoter for the SV40 late genes) a discrete element overlying the startsite itself helps to fix the place of initiation. Additional promoterelements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30 to 110 bp upstream of thestart site, although a number of promoters have been shown to containfunctional elements downstream of the start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the DNA and promotes expression ofthe encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with anenhancer.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Similarly, an enhancer maybe one naturally associated with a nucleic acid sequence, located eitherdownstream or upstream of that sequence. Alternatively, certainadvantages will be gained by positioning the coding nucleic acid segmentunder the control of a recombinant or heterologous promoter, whichrefers to a promoter that is not normally associated with a nucleic acidsequence in its natural environment. A recombinant or heterologousenhancer refers to an enhancer not normally associated with a nucleicacid sequence in its natural environment. Such promoters or enhancersmay include promoters or enhancers of other genes, and promoters orenhancers isolated from any other virus, or prokaryotic or eukaryoticcell, and promoters or enhancers not “naturally occurring,” i.e.,containing different elements of different transcriptional regulatoryregions, and/or mutations that alter expression. For example, promotersthat are most commonly used in recombinant DNA construction include theβ lactamase (penicillinase), lactose and tryptophan (trp) promotersystems. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202 and 5,928,906). Furthermore, it is contemplated that controlsequences, which direct transcription and/or expression of sequenceswithin non-nuclear organelles such as mitochondria, chloroplasts, andthe like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in theorganelle, cell type, tissue, organ, or organism chosen for expression.The promoters employed may be constitutive, tissue-specific, inducible,and/or useful under the appropriate conditions to direct high levelexpression of the introduced DNA segment, such as is advantageous in thelarge-scale production of recombinant proteins and/or peptides. Thepromoter may be heterologous or endogenous.

Additionally any promoter/enhancer combination may be used to driveexpression. Use of a T3, T7 or SP6 cytoplasmic expression system isanother possible embodiment. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct.

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Non-limiting examples of such regions include the human LIMK2 gene(Nomoto et al. (1999) Gene 236(2):259-271), the somatostatin receptor-2gene (Kraus et al., (1998) FEBS Lett. 428(3): 165-170), murineepididymal retinoic acid-binding gene (Lareyre et al., (1999) J. Biol.Chem. 274(12):8282-8290), human CD4 (Zhao-Emonet et al., (1998) Biochem.Biophys. Acta, 1442(2-3):109-119), mouse α-2 (XI) collagen (Tsumaki, etal., (1998), J. Biol. Chem. 273(36):22861-4) D1A dopamine receptor gene(Lee, et al., (1997), DNA Cell Biol. 16(11):1267-1275) insulin-likegrowth factor II (Vu et al., (1997) Biophys Biochem Res. Comm.233(1):221-226) and human platelet endothelial cell adhesion molecule-1(Almendro et al., (1996) J. Immunol. 157(12):5411-5421).

2. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites. IRES elements can be linked toheterologous open reading frames. Multiple open reading frames can betranscribed together, each separated by an IRES, creating polycistronicmessages. By virtue of the IRES element, each open reading frame isaccessible to ribosomes for efficient translation. Multiple genes can beefficiently expressed using a single promoter/enhancer to transcribe asingle message (see U.S. Pat. Nos. 5,925,565 and 5,935,819).

3. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to be more stable and are translated more efficiently. Thus,in other embodiments involving eukaryotes, it is contemplated that theterminator comprise a signal for the cleavage of the RNA, and that theterminator signal promote polyadenylation of the message. The terminatorand/or polyadenylation site elements can serve to enhance message levelsand to minimize read through from the cassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

4. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal or the bovine growth hormone polyadenylationsignal, both of which are convenient and known to function well invarious target cells. Polyadenylation may increase the stability of thetranscript or may facilitate cytoplasmic transport.

5. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with fluorescence activated cell sorting (FACS) analysis.The marker used is not believed to be important, so long as it iscapable of being expressed simultaneously with the nucleic acid encodinga gene product. Further examples of selectable and screenable markersare well known to one of skill in the art.

The vector is introduced to the initially infected cell by suitablemethods. Such methods for nucleic acid delivery for transformation of anorganelle, a cell, a tissue or an organism for use with the currentinvention are believed to include virtually any method by which anucleic acid (e.g., HSV vector) can be introduced into an organelle, acell, a tissue or an organism, as described herein or as would be knownto one of ordinary skill in the art. Non-limiting exemplary methodsinclude: direct delivery of DNA by ex vivo transfection; injection (U.S.Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524,5,702,932, 5,656,610, 5,589,466 and 5,580,859); microinjection (U.S.Pat. No. 5,789,215); electroporation (U.S. Pat. No. 5,384,253); calciumphosphate precipitation; DEAE dextran followed by polyethylene glycol;direct sonic loading; liposome mediated transfection; receptor-mediatedtransfection; microprojectile bombardment (PCT Application Nos. WO94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055,5,550,318, 5,538,877 and 5,538,880); agitation with silicon carbidefibers (U.S. Pat. Nos. 5,302,523 and 5,464,765); Agrobacterium mediatedtransformation (U.S. Pat. Nos. 5,591,616 and 5,563,055); PEG mediatedtransformation of protoplasts (U.S. Pat. Nos. 4,684,611 and 4,952,500);desiccation/inhibition mediated DNA uptake, and any combination of thesemethods, or other methods known to persons of skill in the art. Thecomposition can also be delivered to a cell in a mammal by administeringit systemically, such as intravenously, in a pharmaceutically acceptableexcipient.

B. Methods of DNA Vector Delivery to Cells

1. Ex Vivo Transformation

Methods for transfecting cells and tissues removed from an organism inan ex vivo setting are known to those of skill in the art. Thus, it iscontemplated in the present invention that cells or tissues may beremoved and transfected ex vivo using the nucleic acids and compositionsdescribed herein. In particular aspects, the transplanted cells ortissues may be placed into an organism. In some embodiments, a nucleicacid is expressed in the transplanted cell or tissue.

2. Injection

In certain embodiments, a nucleic acid may be delivered to an organelle,a cell, a tissue or an organism via one or more injections (i.e., aneedle injection), such as, for example, subcutaneously, intradermally,intramuscularly, intravenously, intraperitoneally, etc. Methods ofinjection are well known to those of ordinary skill in the art (e.g.,injection of a composition comprising a saline solution). Furtherembodiments of the present invention include the introduction of anucleic acid by direct microinjection. The amount of composition of thepresent invention used may vary upon the nature of the cell, tissue ororganism affected.

3. Electroporation

In certain embodiments of the present invention, a nucleic acid isintroduced into an organelle, a cell, a tissue or an organism viaelectroporation. Electroporation involves the exposure of a suspensionof cells and DNA to a high voltage electric discharge. In some variantsof this method, certain cell wall degrading enzymes, such as pectindegrading enzymes, are employed to render the target recipient cellsmore susceptible to transformation by electroporation than untreatedcells (U.S. Pat. No. 5,384,253). Alternatively, recipient cells can bemade more susceptible to transformation by mechanical wounding.

4. Liposome Mediated Transfection

In a further embodiment of the invention, a composition as describedherein, such as a vector having a modified ICP10 polynucleotide, may beentrapped in a lipid complex such as, for example, a liposome. Liposomesare vesicular structures characterized by a phospholipid bilayermembrane and an inner aqueous medium. Multilamellar liposomes havemultiple lipid layers separated by aqueous medium. They formspontaneously when phospholipids are suspended in an excess of aqueoussolution. The lipid components undergo self rearrangement before theformation of closed structures and entrap water and dissolved solutesbetween the lipid bilayers. Also contemplated is an nucleic acidcomplexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen).

In certain embodiments of the invention, a liposome may be complexedwith a hemagglutinatin virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry of liposomeencapsulated DNA (Kaneda et al., (1989) Science 20; 243(4889):375-8). Inother embodiments, a liposome may be complexed or employed inconjunction with nuclear non histone chromosomal proteins (HMG1) (Katoet al., (1991) J Biol Chem. (1991) February 25; 266(6):3361-4). In yetfurther embodiments, a liposome may be complexed or employed inconjunction with both HVJ and HMG 1. In other embodiments, a deliveryvehicle may comprise a ligand and a liposome.

5. Receptor Mediated Transfection

A nucleic acid may be delivered to a target cell via receptor mediateddelivery vehicles. This approach takes advantage of the selective uptakeof macromolecules by receptor mediated endocytosis. In view of the celltype specific distribution of various receptors, this delivery methodadds another degree of specificity to the present invention.

In certain embodiments, the receptor mediated gene targeting vehiclecomprises a receptor specific ligand and a nucleic acid binding agent.Other embodiments comprise a receptor specific ligand to which thenucleic acid to be delivered has been operatively attached. Severalligands have been used for receptor mediated gene transfer including theepidermal growth factor (EGF), which has been used to deliver genes tosquamous carcinoma cells as described in European Patent No. EPO 0 273085.

In other embodiments, a nucleic acid delivery vehicle component of acell specific nucleic acid targeting vehicle may comprise a specificbinding ligand in combination with a liposome. The nucleic acid(s) to bedelivered are housed within the liposome and the specific binding ligandis functionally incorporated into the liposome membrane. The liposomewill thus specifically bind to the receptor(s) of a target cell anddeliver the contents to a cell.

In still further embodiments, the nucleic acid delivery vehiclecomponent of a targeted delivery vehicle may be a liposome itself, whichwill preferably comprise one or more lipids or glycoproteins that directcell specific binding. For example, lactosyl ceramide, a galactoseterminal asialganglioside, has been incorporated into liposomes and anincrease in the uptake of the insulin gene by hepatocytes has beenobserved (Nicolau et al., (1987) Methods Enzymol. 149:157-76). It iscontemplated that the tissue specific transforming constructs of thepresent invention can be specifically delivered into a target cell in asimilar manner.

6. Microprojectile Bombardment

Microprojectile bombardment techniques can be used to introduce anucleic acid into at least one, organelle, cell, tissue or organism(U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No.5,610,042; and PCT Application No. WO 94/09699). This method depends onthe ability to accelerate microprojectiles that are either coated withDNA or contain DNA, to a high velocity allowing them to pierce cellmembranes and enter cells without killing them. The microprojectiles maybe comprised of any biologically inert substance, such as tungsten,platinum, or gold. For the bombardment, cells in suspension areconcentrated on filters or solid culture medium. Alternatively, immatureembryos or other target cells may be arranged on solid culture medium.The cells to be bombarded are positioned at an appropriate distancebelow the microprojectile bombardment device on a stopping plate. A widevariety of microprojectile bombardment techniques useful for practicewith the current invention will be known to persons of skill in the art.

C. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organism that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny. Asused herein, the terms “engineered” and “recombinant” cells or hostcells are intended to refer to a cell into which an exogenous nucleicacid sequence, such as, for example, a vector, has been introduced.Therefore, recombinant cells are distinguishable from naturallyoccurring cells that do not contain a recombinantly introduced nucleicacid.

A tissue may comprise a host cell or cells to be transformed with a cellmembrane fusion-generating HSV-2 mutant. The tissue may be part orseparated from an organism. In certain embodiments, a tissue maycomprise, but is not limited to, adipocytes, alveolar, ameloblasts,neural, basal cells, blood (e.g., lymphocytes), blood vessel, bone, bonemarrow, glial cell, breast, cartilage, cervix, colon, cornea, embryonic,endometrium, endothelial, epithelial, esophagus, facia, fibroblast,follicular, ganglion cells, glial cells, goblet cells, kidney, liver,lung, lymph node, muscle, neuron, ovaries, pancreas, peripheral blood,prostate, skin, small intestine, spleen, stem cell, stomach, testes, andall cancers thereof.

In certain embodiments, the host cell or tissue may be comprised in atleast one organism. In certain embodiments, the organism may be, but isnot limited to, a prokaryote (e.g., a eubacteria, an archaea) or aeukaryote, as would be understood by one of ordinary skill in the art.

Numerous cell lines and cultures are available for use as a host cell,and are commercially available through organizations such as theAmerican Type Culture Collection (ATCC). An appropriate host can bedetermined by one of skill in the art based on the vector backbone andthe desired result. Exemplary non-limiting cell types available forvector replication and/or expression include bacteria, such as E. coli(e.g., E. coli strains RR1, LE392, B, X 1776 (ATCC No. 31537), W3110, F,lambda, DH5α, JM109, and KC8); bacilli e.g., Bacillus subtilis; otherenterobacteriaceae e.g., Salmonella typhimurium, Serratia marcescens, aswell as a number of commercially available bacterial hosts and competentcells such as SURE® Competent Cells and SOLOPACK™ Gold Cells(STRATAGENE®, La Jolla, Calif.). Non-limiting examples of eukaryotichost cells for replication and/or expression of a vector include, HeLa,NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

D. Viral Vector Packaging and Propagation.

1. Viral Packaging

In specific embodiments of the present invention, after the ICP10 genehas been modified, it is inserted into the virus through homologousrecombination. Typically, this is done by co-transfecting the plasmidDNA containing the modified ICP10 gene with purified HSV-2 genomic DNAinto Vero cells using Lipofectamine. The recombinant virus is thenidentified (typically by screening the virus plaques for the presence ofa selectable marker) and selecting plaques containing the modified ICP10polynucleotide. The selected recombinant virus is then characterized invitro to confirm that the modified ICP10 gene has been correctlyinserted into the HSV-2 genome to replace the original ICP10 gene. Viralpackaging and in vitro characterization are described in more detail inExamples 1 and 2.

2. Preparation of Viral Stocks

Once the recombinant HSV-2 mutant virus has been selected, viral stocksare prepared as follows. Vero cells are grown in 10% fetal bovine serum(FBS) and infected with 0.01 plaque forming units (pfu) per cell.Viruses are then harvested from the cells 2 days later by repeatedfreezing and thawing and sonication. The harvested virus is thenpurified as described (Nakamori, et al., (2003) Clinical Cancer Res.9(7):2727-2733). The purified virus is then titered (as described inExample 10), aliquoted and stored at −80° C. until use.

E. Protein Expression Systems

Protein expression systems may be utilized in the generation of DNAvector compositions of the present invention for example, to express thepolypeptide encoded by the modified ICP10 polynucleotide for functionalstudies. Numerous expression systems exist that comprise at least a partor all of the compositions discussed above. Prokaryote- and/oreukaryote-based systems can be employed for use with the presentinvention to produce nucleic acid sequences, or their cognatepolypeptides, proteins and peptides. Many such systems are commerciallyand widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986 and 4,879,236 and is commercially available(e.g., CLONTECH, Inc. Mountain View, Calif.).

Other examples of commercially available expression systems include aninducible mammalian expression system, which involves a syntheticecdysone-inducible receptor, or a pET expression system, or an E. coliexpression system (STRATAGENE, LaJolla, Calif.); Atetracycline-regulated expression system, an inducible mammalianexpression system that uses the full-length CMV promoter or a yeastexpression system designed for high-level production of recombinantproteins in the methylotrophic yeast Pichia methanolica (INVITROGEN,Carlsbad, Calif.).

It is contemplated that the proteins, polypeptides or peptides producedby the methods of the invention may be “overexpressed”, i.e., expressedin increased levels relative to its natural expression in cells. Suchoverexpression may be assessed by a variety of methods, includingradio-labeling and/or protein purification. However, simple and directmethods are preferred, for example, those involving SDS/PAGE and proteinstaining or western blotting, followed by quantitative analysis, such asdensitometric scanning of the resultant gel or blot. A specific increasein the level of the recombinant protein, polypeptide or peptide incomparison to the level in natural cells is indicative ofoverexpression, as is a relative abundance of the specific protein,polypeptides or peptides in relation to the other proteins produced bythe host cell and, e.g., visible on a gel.

V. Functional Roles of a HSV-2 Mutant

A HSV-2 mutant as described herein displays multiple functional roles asan oncolytic agent. For example, the virus can destroy tumor cells bylysis, as well as by syncytial formation, and induction of apoptosis inboth infected cells as well as by-stander cells. Furthermore, tumordestruction by the HSV-2 mutant induces a potent anti-tumor immuneresponse that further contributes to the therapeutic efficacy of themutant virus as an oncolytic agent for the treatment of malignantdisease.

The HSV-2 mutant virus displays selective replication in cycling, butnot non-cycling cells. As described in more detail in Example 4, themutant HSV-2, lacking protein kinase activity, shows at least a 40-folddecrease in growth in non-cycling cells as compared to growth in cyclingcells. In contrast, the wild-type HSV-2 is only marginally affected inits growth characteristics between cycling and non-cycling cells.Therefore, the HSV-2 mutant as described herein is well suited for useas an oncolytic agent in cycling cells having an activated Ras pathway,such as tumor cells.

The modified HSV-2 described herein has superior tumor cell killingability compared to other oncolytic viruses and the wild-type HSV-2.Using an in vitro assay as described in Example 5, demonstrates that thekilling ability of FusOn-H2 against human tumor cells of differenttissue origins is significantly stronger than that of the oncolyticHSV-1 described in U.S. patent application Ser. No. 10/397,635 and/ortested until today, and even exceeds that of the parental wild-typeHSV-2. Furthermore, as described in the Example 6, a single injection ofthe virus of the present invention at a moderate dose (1×10⁶plaque-forming-unit) led to the complete disappearance of breast tumororthotopically established in nude mice in 100% of the animals (n=8),while administration of the same dose of oncolytic HSV-1 only shrank thetumor in less than 30% of the mice.

In addition to the lytic and fusogenic activities, the HSV-2 mutant alsohas potent apoptotic inducing activity and is capable of inducing apotent anti-tumor immune response. In an in vitro setting, the HSV-2mutant can induce apoptosis in cells infected with the virus as well asnon-infected by-stander cells that surround the infected cells.Furthermore, HSV-2 mutant is effective at inducing apoptosis of tumorcells in vivo. This is described in greater detail in Example 8. Notonly are the compositions described herein more effective at killingtumor cells than other oncolytic viruses, the HSV-2 mutant displays astrong therapeutic effect against primary and metastatic tumor in vivoby induction of a strong anti-tumor immune response. As described inExample 9, the adoptive transferred CTL from FusOn-H2 treated mice caninhibit the growth of the original tumor and effectively prevent themetastases developing.

Apoptosis, or programmed cell death, is an essential process for normalembryonic development, maintaining homeostasis in adult tissues, andsuppressing carcinogenesis. In some embodiments of the invention, themodified HSV-2 is a potent inducer of apoptosis in tumor cells infectedwith the virus, and in non-infected by-stander tumor cells. For example,in a particular embodiment tumor cells were infected with an HSV-2construct in which parts of the protein kinase domain of the ICP10 genewas replaced with a gene encoding the green fluorescent protein (GFP).Infected cells could be identified under a fluorescent microscope byvisualizing the GFP, and cells undergoing apoptosis were identified asevidenced by their chromatin condensation. The ratio of cells showingchromatin condensation to GFP expression was 2.6:1, suggesting thatthere was a substantial number of tumor cells undergoing apoptosis, thatwere not infected with the modified HSV-2. The ability of the oncolyticvirus of the present invention to induce apoptosis is described in moredetail in Example 8.

Strong anti-tumor immune responses are useful in combating malignantdisease. The HSV-2 mutant described herein is capable of inducing apotent antitumor immune response against primary and metastatic tumorsin vivo. In a particular embodiment, the mutant HSV-2 (FusOn-H2)selectively replicated in and lysed tumor cells in a mouse mammary tumormodel using the 4T1 mouse mammary tumor cell line, and showed a strongtherapeutic effect against primary and metastatic tumor in vivo byinduction of strong antitumor immune response. Specifically, adoptivetransferred cytotoxic T lymphocytes (CTL) from FusOn-H2 treated mice caninhibit growth of the original tumor and effectively prevent metastasisin mice not treated with FusOn-H2. This is described in more detail inExample 9.

VI. Pharmaceutical Compositions and Routes of Administration

A. General Considerations

Compositions of the present invention can be administered as apharmaceutical composition comprising either a recombinant HSV-2 mutanthaving a modified ICP10 gene, or as a naked (non-viral) DNA vectorhaving a modified ICP10 gene, as described herein. The compositions ofthe present invention include classic pharmaceutical preparations. Ingeneral, the compositions of the present invention can be administeredas pharmacological agents by dissolving or dispersing the composition ina pharmaceutically acceptable carrier or aqueous medium. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the compositions of the invention, its use in atherapeutic composition is contemplated. Supplementary activeingredients, such as other anti-disease agents, can also be incorporatedinto the pharmaceutical composition. Administration of the compositionwill be via any common route so long as the target cell is available viathat route. Exemplary administration routes include oral, nasal, buccal,rectal, vaginal or topical. Alternatively, administration may be byorthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal,intravenous, or direct intratumoral injection. The pharmaceuticalformulations, dosages and routes of administration for the compositionsof the present invention are described infra.

B. Pharmaceutical Formulation of HSV-2 Mutant

The mutant viral composition of the present invention can be prepared asa pharmacologically acceptable formulation. Typically, the mutant virusis mixed with an excipient which is pharmaceutically acceptable andcompatible with the virus. Suitable excipients are, for example, water,saline, dextrose, glycerol, ethanol, or the like and combinationsthereof. In addition, if desired, the preparation may contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH-buffering agents, adjuvants or immunopotentiators, which enhance theeffectiveness of the viral mutant (See, Remington's PharmaceuticalSciences, Gennaro, A. R. et al., eds., Mack Publishing Co., pub., 18thed., 1990). For example, a typical pharmaceutically acceptable carrierfor injection purposes may comprise from 50 mg up to about 100 mg ofhuman serum albumin per milliliter of phosphate buffered saline.Additional non-limiting exemplary non-aqueous solvents suitable for usein the formulation of a pharmacologically acceptable composition includepropylene glycol, polyethylene glycol, vegetable oil, sesame oil, peanutoil and injectable organic esters such as ethyloleate. Exemplarynon-limiting aqueous carriers include water, aqueous solutions, salinesolutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Determining the pH and exact concentration of the variouscomponents of the pharmaceutical composition is routine and within theknowledge of one of ordinary skill in the art (See Goodman and Gilman'sThe Pharmacological Basis for Therapeutics, Gilman, A. G. et al., eds.,Pergamon Press, pub., 8th ed., 1990).

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother sterile ingredients as required and described above. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients as described above.

C. Routes and Dosages for Administration of RSV-2 Mutant

The mutant viral composition may be delivered by any route that providesaccess to the target tissue. Exemplary non-limiting routes ofadministration may include oral, nasal, buccal, rectal, vaginal topical,or by injection (including orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal, intravenous, or direct intratumoralinjection). Typically, the viral mutant would be prepared as aninjectable, either as a liquid solution or a suspension; a solid formsuitable for solution in, or suspension in, liquid prior to injectionmay also be prepared. The preparation also may be emulsified.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermolysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.

Those of skill in the art will recognize that the best treatmentregimens for using a composition of the present invention to providetherapy can be straightforwardly determined. This is not a question ofexperimentation, but rather one of optimization, which is routinelyconducted in the medical arts. For example, in vivo studies in miceprovide a starting point from which to begin to optimize the dosage anddelivery regimes. The frequency of injection may initially be once aweek. However, this frequency might be optimally adjusted from one dayto every two weeks to monthly, depending upon the results obtained fromthe initial clinical trials and the needs of a particular patient. Humandosage amounts can initially be determined by extrapolating from theamount of composition used in mice.

1. Dosages

The amount of viral vector delivered will depend on several factorsincluding number of treatments, subject to be treated, capacity of thesubjects immune system to synthesize anti-viral antibodies, the targettissue to be destroyed, and the degree of protection desired. Theprecise amount of viral composition to be administered depends on thejudgment of the practitioner and is peculiar to each individual.However, suitable dosage ranges from 10⁵ plaque forming units (pfu) to10¹⁰ pfu. In certain embodiments, the dosage of viral DNA may be about10⁵, 10⁶, 10⁷, 10⁸, 10⁹, up to and including 10¹⁰ pfu.

D. Non-viral DNA Vector Formulation

In addition to the formulations described above for viral pharmaceuticalformulation, the non-viral DNA vector can also be prepared as a sterilepowder for the preparation of pharmacologically acceptable sterilesolutions. Typical methods for preparation of sterile powder includevacuum-drying and freeze-drying techniques which yield a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

E. Routes and Dosages for Administration of Non-Viral DNA Vector

Several methods for the delivery of non-viral vectors for the transferof a polynucleotide of the present invention into a mammalian cell iscontemplated. These include calcium phosphate precipitation,DEAE-dextran, electroporation, direct microinjection, DNA-loadedliposomes and lipofectamine-DNA complexes, cell sonication, genebombardment using high velocity microprojectiles, and receptor-mediatedtransfection as discussed previously. Some of these techniques may besuccessfully adapted for in vivo or ex vivo use.

In some embodiments of the present invention, the expression vector maysimply consist of naked recombinant DNA or plasmids comprising thepolynucleotide. Transfer of the construct may be performed by any of themethods mentioned herein which physically or chemically permeabilize thecell membrane. This is particularly applicable for transfer in vitro,but it may be applied to in vivo use as well.

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al., employed lactosyl-ceramide, agalactose-terminal asialganglioside, incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes(Nicolau et al., (1987) Methods Enzymol. 149:157-76). Thus, it isfeasible that a nucleic acid encoding a particular gene also may bespecifically delivered into a cell type by any number of receptor-ligandsystems with or without liposomes. For example, epidermal growth factor(EGF) may be used as the receptor for mediated delivery of a nucleicacid into cells that exhibit upregulation of EGF receptor (as describedin European Patent No. EP 0 273 085) and mannose can be used to targetthe mannose receptor on liver cells.

In certain embodiments, DNA transfer may more easily be performed underex vivo conditions. Ex vivo gene therapy refers to the isolation ofcells from an animal, the delivery of a nucleic acid into the cells invitro, and then the return of the modified cells back into an animal.This may involve the surgical removal of tissue/organs from an animal orthe primary culture of cells and tissue.

1. Dosages

In certain embodiments it is envisioned that the dosage may vary frombetween about 10³ pfu/kg body weight to about 10⁸ pfu/kg body weight. Incertain embodiments, the dosage may be from about 10³, 10⁴, 10⁵, 10⁶,10⁷, up to and including 10⁸ pfu/kg body weight. Of course, this dosageamount may be adjusted upward or downward, as is routinely done in suchtreatment protocols, depending on the results of the initial clinicaltrials and the needs of a particular patient.

VI. Combination Treatments

In order to increase the effectiveness of the methods and compositionsof the present invention, it may be desirable to combine the methods andcompositions disclosed herein with other anti-cancer agents. Thisprocess may involve contacting the cancer cell with a composition of thepresent invention in conjunction with at least one other anti-canceragent. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations.Where two distinct formulations are used, the cancer cell may becontacted either by both formulations at the same time, or where oneformulation precedes the other (e.g. where a composition of the presentinvention is administered either preceding or following administrationof another anti-cancer agent) or any combination or repetitive cyclethereof. In embodiments where a composition of the present invention andthe other agent are administered separately, one would generally ensurethat a significant period of time did not expire between the time ofeach delivery, such that the composition of the present invention andthe other agent would still be able to exert an advantageously combinedeffect on the cancer cell. This time interval between administration ofthe two formulations may range from minutes to weeks.

Non-limiting examples of anti-cancer agents that may be used inconjunction with the compositions or methods of the present inventionmay include chemotherapeutic agents (e.g., cisplatin (CDDP),carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, ifosfamide, melphalan, chlorambucil, busulfan,nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin,plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogenreceptor binding agents, taxol, gemcitabien, navelbine, farnesyl-proteintransferase inhibitors, transplatinum, 5-fluorouracil, vincristin,vinblastin and methotrexate, or any analog or derivative variant of theforegoing); radio-therapeutic agents (e.g., γ-rays, X-rays, microwavesand UV-irradiation, and/or the directed delivery of radioisotopes totumor cells); immunotherapeutic and immunomodulatory agents; genetherapeutic agents; pro-apoptotic agents and other cell cycle regulatingagents well known to persons of skill in the art.

Immunotherapy can also be used in conjunction with the compositions andmethods described herein as a combination therapy for the treatment ofmalignant disease. Immunotherapeutics generally rely on the use ofimmune effector cells and molecules to target and destroy cancer cells.The immune effector may be, for example, an antibody specific for somemarker on the surface of a tumor cell. The antibody alone may serve asan effector of therapy or it may recruit other cells (e.g. cytotoxicT-cells or NK cells) to actually effect cell killing. The antibody alsomay be conjugated to a drug or toxin (e.g., a chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. In some embodiments, the effector maybe a lymphocyte carrying a surface molecule that interacts, eitherdirectly or indirectly, with a tumor cell target. In other embodiments,the tumor cell must bear some marker that is amenable to targeting.Non-limiting exemplary tumor markers suitable for targeting may includecarcinoembryonic antigen (CEA), prostate specific antigen, urinary tumorassociated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG,Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B and p155.

Gene therapy can also be used in conjunction with the compositions andmethods described herein as a combination therapy for the treatment ofmalignant disease. Gene therapy as a combination treatment relies on thedelivery and expression of a therapeutic gene, separate from the mutantHSV-2 described herein. The gene therapy can be administered eitherbefore, after, or at the same time as the HSV-2 mutant described herein.Exemplary non-limiting targets of gene therapy include immunomodulatoryagents, agents that affect the up regulation of cell surface receptorsand GAP junctions, cytostatic and differentiation agents, inhibitors ofcell adhesion, or agents that induce or increase the sensitivity oftarget cells to apoptosis. Exemplary non-limiting immunomodulatory genesthat can be used as part of gene therapy in combination with the presentinvention include tumor necrosis factor; interferon alpha, beta, andgamma; IL-2 and other cytokines; F42K and other cytokine analogs; OrMIP-1, MIP-1 beta, MCP-1, RANTES, and other chemokines.

An exemplary inhibitor of cellular proliferation is p16. The majortransitions of the eukaryotic cell cycle are triggered bycyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4(CDK4), regulates progression through the G1. The activity of thisenzyme may be to phosphorylate Rb at late G1. The activity of CDK4 iscontrolled by an activating subunit, D-type cyclin, and by an inhibitorysubunit, the p16INK4 that specifically binds to and inhibits CDK4, andthus may regulate Rb phosphorylation. The p16INK4 gene belongs to anewly described class of CDK-inhibitory proteins that also includesp16B, p19, p21WAF1, and p27KIP1. Homozygous deletions and mutations ofthe p16INK4 gene are frequent in human tumor cell lines. Since thep16INK4 protein is a CDK4 inhibitor deletion of this gene may increasethe activity of CDK4, resulting in hyperphosphorylation of the Rbprotein. Other genes that may be employed with gene therapy to inhibitcellular proliferation include Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I,MEN-II, zac1, p73, VHL, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16fusions, p21/p27 fusions, anti-thrombotic genes (e.g., COX-1, TFPI),PGS, Dp, E2F, ras, myc, neu, raf, erb, frns, trk, ret, gsp, hst, abl,E1A, p300, genes involved in angiogenesis (e.g., VEGF, FGF,thrombospondin, BAI-1, GDAIF, or their receptors) and MCC.

It is further contemplated that the up regulation of cell surfacereceptors or their ligands such as Fas/Fas ligand, DR4 or DR5/TRAILwould potentiate the apoptotic inducing abilities of the presentinvention by establishment of an autocrine or paracrine effect onhyperproliferative cells. Increases in intercellular signaling byelevating the number of GAP junctions would increase theanti-hyperproliferative effects on a neighboring hyperproliferative cellpopulation. In other embodiments, cytostatic or differentiation agentscan be used in combination with the present invention to improve theanti-hyperproliferative efficacy of the treatments. Inhibitors of celladhesion are contemplated to improve the efficacy of the presentinvention. Examples of cell adhesion inhibitors are focal adhesionkinase (FAKs) inhibitors and Lovastatin. It is further contemplated thatother agents that increase the sensitivity of a hyperproliferative cellto apoptosis, such as the antibody c225, could be used in combinationwith the present invention to improve the treatment efficacy.

Hormonal therapy may also be used in conjunction with the presentinvention. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen. This treatment is often used in combinationwith at least one other cancer therapy as a treatment option or toreduce the risk of metastases.

All patents, patent applications, and other publications cited in thisapplication, including published amino acid or polynucleotide sequences,are incorporated by reference in their entirety for all purposes.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Construction of FusOn-H2

The construction of the exemplary FusOn-H2 is illustrated in FIG. 1.Initially the HSV genome region comprising the ICP10 left-flankingregion (equivalent to nucleotide number of HSV-2 genome 85994-86999) wasamplified with the following exemplary pair of primers:5′-TTGGTCTTCACCTACCGACA (SEQ ID NO:1); and 3′-GACGCGATGAACGGAAAC (SEQ IDNO:2). The RR domain and the right-flank region (equivalent to thenucleotide sequence number of HSV-2 genome 88228-89347) were amplifiedwith the following exemplary pair of primers: 5′-ACACGCCCTATCATCTGAGG(SEQ ID NO:13); and 5′-AACATGATGAAGGGGCTTCC (SEQ ID NO:14). These twoPCR products were cloned into pNeb 193 through EcoRI-NotI-XbaI ligationto generate pNeb-ICP10-deltaPK. Then, the DNA sequence containing theCMV promoter-EGFP gene was PCR amplified from pSZ-EGFP with thefollowing exemplary pair of primers: 5′-ATGGTGAGCAAGGGCGAG (SEQ IDNO:3); and 3′-CTTGTACAGCTCGTCCATGC (SEQ ID NO:4). The PCR-amplified DNAwas then cloned into the deleted PK locus of pNeb-ICP10-deltaPK throughBglII and NotI ligation to generate pNeb-PKF-2. During the design of PCRamplification strategies, the primers were designed such that the EGFPgene was fused in frame with the remaining RR domain of the ICP10 geneso that the new protein product of this fusion gene comprises the intactfunctional EGFP, which would facilitate the selection of the recombinantvirus in the following experimental steps.

The modified ICP10 gene was inserted into the virus through homologousrecombination by co-transfecting the pNeb-PKF-2 plasmid DNA withpurified HSV-2 genomic DNA (strain 186) into Vero cells bylipofectamine. The recombinant virus was screened and identified byselecting GFP-positive virus plaques. During the screening process, itwas noticed that all of the GFP-positive plaques showed clear syncytialformation of the infected cells, indicating that this modified virusinduces widespread cell membrane fusion, in specific embodiments of theinvention. A total of 6 plaques were picked. One of them, referred to asFusOn-H2, was selected for further characterization and for all of thesubsequent experiments.

Example 2 In Vitro Characterization

The exemplary FusOn-H2 vector was characterized by standard methods inthe art.

Southern Blot Analysis.

To confirm that the modified ICP10 gene has been correctly inserted intothe HSV-2 genome to replace the original ICP10 gene, virion DNA wasextracted from purified FusOn-H2 virus stock. As a control, virion DNAfrom the parental wild type HSV-2 was extracted according to the sameprocedure. The virion DNA was digested with BamHI and electrophoresed inan 0.8% agarose gel. BamHI digestion generates an 7390 bp DNA fragmentfrom the wild type HSV-2 genome that comprises the entire ICP10 gene andits left and right flank regions. However, digestion of FusOn-H2 genomeby the same enzyme generates two DNA fragments from the ICP10 genelocus: 1) a 4830 bp fragment comprising the left-flank and the CMVpromoter sequence; and 2) a 3034 bp sequence comprising the GFP, RR, andthe right-flank region. The DNA was transferred to a nylon membrane andhybridized with four probes prepared from: 1) the left-flank region ofDNA sequence, 2) the whole PK region; 3) the right-flank region of PK;and 4) GFP gene. The result (FIG. 2) showed that all of the probesexcept the one made from GFP gene hybridized to a 7390 hp DNA band. Theleft-flank probe hybridized to a DNA band that was identified by theprobes prepared from the GFP and the right-flank DNA sequences. Theprobe made from the PK domain sequence failed to hybridized to either ofthe DNA fragments, indicating that the PK domain has been completelydeleted from the genome of FusOn-H2.

Western Blot Hybridization.

To further confirm the correctness of the modified ICP10 gene in thegenome of FusOn-H2, proteins were extracted from Vero cells infectedwith either FusOn-H2 or the parental wild type HSV-2, or from cellstransfected with the pSZ-EGFP plasmid DNA. The proteins were separatedon a 12% SDS-PAGE gel and transferred to Hybond-C membrane. The membranewas then blotted with an anti-GFP monoclonal antibody (Anti-GFP #Ab290,ABCAM Inc., Cambridge, Mass.). This anti-GFP antibody picked up thesmaller GFP protein (around 28 kD) expressed from the pSZ-EGPtransfected cells. The same antibody also identified a significantlybigger protein band (the size of the fusion protein is expected to bearound 120 kD). However, this antibody failed to react to any proteinproducts from wild type HSV-2 infected cells, confirming the specificityof this antibody. These results further confirm that the GFP gene hasbeen correctly fused with the remaining RR domain of the ICP10 gene inthe FusOn-H2 genome.

Example 3 In Vitro Phenotypic Characterization of FusOn-H2

To determine the phenotype of FusOn-H2, the present inventors infectedVero cells with either wild type HSV-2 or FusOn-H2, or the cells wereleft uninfected. Twenty-four hours after infection, a clear syncytialformation was visible in the cell monolayer infected with FusOn-H2. Nosyncytium was seen in either uninfected cells or cells infected with thewild type HSV-2. Similar syncytial formation was also observed in humantumor cells of different tissue origin. In some tumor cells, theinfection of wild type HSV-2 also induced some syncytial formation.However, the syncytial formation induced by FusOn-H2 on these cellsusually was significantly more profound. So in this case, the FusOn-H2has an enhanced fusogenic activity when compared with the parental wildtype HSV-2. These results indicate that FusOn-H2 is phenotypicallydifferent from the parental virus in that its infection induceswidespread syncytial formation or enhances the intensity of syncytialformation in tumor cells. Neither the PK domain nor the entire ICP10gene have been previously reported to have any functional link with cellmembrane fusion (Smith et al., (1998) J. Virol. 72(11):9131-9141; Smithet al., (1994) Virol 200(2):598-612; Smith et al., (1992) J. Gen. Virol.73(pt6):1417-1428). In some embodiments, the addition of the GFP geneand/or the replacement of the natural promoter of ICP10 with the strongCMV promoter contributed to this phenotypic change of the virus. Thefusogenic phenotype of FusOn-H2 is important for the application ofoncolytic purposes, since syncytial formation induced by a type 1oncolytic HSV was shown to significantly increase the killing ability ofthe virus against human tumor cells, for example (U.S. patentapplication Ser. No. 10/397,635, filed Mar. 26, 2003).

Example 4 Growth Curve of FusOn-H2 in Cycling and Non-Cycling Cells

To determine the property of selective replication of FusOn-H2 individing (tumor) cells, the inventors infected Vero cells, cultured inmedium containing either 10% fetal bovine serum(FBS) (cells therefore infully cycling) or in medium containing no FBS (cells in non-cycling),with either wild type HSV-2 or FusOn-H2. Virus was harvested atdifferent time points after infection and titrated with Vero cells. Thegrowth of wild type virus was only marginally affected (less than2-fold) when the cells were put in a non-cycling state. In contrast, thegrowth of FusOn-H2 in non-cycling cells was dramatically reduced (morethan 40-fold) when compared with the virus yield from cells in a cyclingstate. These results (FIG. 5) indicated that, although fully replicationcompetent in tumor cells, the FusOn-H2 has minimal replicationcapability in non-cycling cells, which usually represent the normalsomatic cells in the body, thus providing for selective replicationcapability of FusOn-H2 in tumor cells.

Example 5 In Vitro Killing Assay of FusOn-H2 Against Human Tumor Cells

Next, the present inventors directly compared the in vitro oncolyticeffect of FusOn-H2 and its parental wild type HSV-2 or an oncolyticvirus constructed from the type 1 HSV (HSV-1). Exemplary human ovariancancer cell line Skov-3 or human breast cancer cell line MDA-MB-435 wereinfected with the viruses at either 0.01 or 0.1 pfu/cell, and the cellviabilities were determined by calorimetric lactate dehydrogenase (LDH)assay, for example, at either 24 or 48 h after virus infection. Theresult (FIG. 6) demonstrates that, among the oncolytic HSVs tested, theFusOn-H2 has the highest killing ability against both human tumor celllines. Its killing ability was even significantly higher than that fromits parental wild-type virus due to its ability to induce syncytialformation in the tumor cells.

Example 6 In Vivo Therapeutic Evaluation of FusOn-H2

The exemplary FusOn-H2 virus was characterized under in vivo conditions.

Against Human Breast Cancer Xenografts

To evaluate the anti-tumor effect of FusOn-H2 in vivo, the presentinventors injected the virus at a very moderate dose (1×10⁶ pfu)directly into established xenografts (around 5-8 mm in diameter) ofhuman breast cancer (from implantation of MDA-MB-435 cells in themammary fat pad). For comparison purposes, the present inventorsincluded an oncolytic HSV derived from HSV-1 (Baco-1), that was used atthe same dose as FusOn-H2; Baco-1 is described in U.S. patentapplication Ser. No. 10/397,635, filed Mar. 26, 2003. Tumor sizes weremeasured weekly for 4 weeks. As compared with the PBS controls, a singleinjection of either viruses had an immediate effect on tumor growth(FIG. 7). Within 1 week of virus injection, the tumors in mice treatedwith either of the oncolytic viruses were significantly smaller thantumors injected with PBS (P<0.001). From week 2 to week 4, however,FusOn-H2 produced significantly greater anti-tumor effects than didBaco-1 (P<0.01). All of the animals (8 of 8) were tumor-free by week 2after FusOn-H2 administration. By contrast, only 2 mice in the groupinjected with Baco-1 were tumor-free. In the other 6 mice, tumors thathad shrunk initially began to re-grow by week 3 after virus injection.These results indicate that FusOn-H2 is a potent anti-tumor agentagainst human breast cancer and is significantly more effective than thefusogenic oncolytic HSV constructed from HSV-1.

Against Human Ovarian Cancer Xenografts

Peritoneal invasion of ovarian cancer is a common and serious clinicalproblem. It has been reported that about 70% of late-stage ovariancancer patients have metastatic disease in the peritoneal cavity. Thepresent inventors therefore chose a peritoneal metastasis model(xenografted Skov-3 cells) as a means to test the efficacy of FusOn-H2against human ovarian cancer, for example. Freshly harvested Skov-3cells were inoculated into the peritoneal cavities of nude mice at adose of 3×10⁶ cells/mouse. Two weeks later, mice received a singleintraperitoneal (i.p.) injection with 3×10⁶ pfu of either Baco-1,FusOn-H2, or PBS (control) at a site distant from that of tumor cellimplantation. This therapeutic injection was repeated one week later.Four weeks after the initial therapeutic injection, mice were euthanizedand the tumor growth in the abdomen cavity was evaluated. There was aclear i.p. dissemination of tumor in either PBS- or Baco-1-treatedgroup, as indicated by the revelation of multiple tumor nodules acrossthe cavity in each animal of these treatment groups (FIG. 8 and Table1).

TABLE 1 Number and weight of tumor nodules in the abdominal cavity afteroncolytic treatment of human ovarian cancer xenografts Treatments PBSBaco-1 FusOn-H2 Mouse Tumor Tumor Tumor Tumor Tumor Tumor no. nodulesweight (g) nodules weight (g) nodules weight (g) 1 8 0.81 5 0.93 1 0.152 12 0.93 1 0.02 0 0 3 9 0.65 12 1.07 0 0 4 14 1 0 0 0 0 5 7 0.48 151.35 0 0 6 30 1.7 2 0.63 0 0 7 19 2.29 9 0.98 0 0 8 25 1.74 4 0.83 0 0mean 15.5 1.20 6 0.72 0.12 0.018 SD 8.4 0.6 5.4 0.4 0.35 0.05

As compared with PBS, Baco-1 treatment provided a certain therapeuticeffect against the established ovarian cancer; one mouse was totallytumor-free and one had significantly-reduced tumor nodule (only onetumor nodule was found). The therapeutic effect, however, of FusOn-H2was clearly more profound. Seven of the eight mice in FusOn-H2-treatedgroup were entirely tumor free by the end of the experiment (Table 1 andFIG. 8). The only mouse that was not tumor-free bore a single tumornodule that was much smaller than those in Baco-1- or PBS-treated mice.These results clearly demonstrate that FusOn-H2 is also extremelyeffective at treating human solid tumors established in a relativelylarge cavity and even when the virus was administered at a very moderatedose.

Example 7 In Vivo Toxicity Evaluation of FusOn-H2

As a first step toward evaluating the toxicity of FusOn-H2, the presentinventors injected either wild type HSV-1, HSV-2 or FusOn-H2 at 5×10⁶pfu subcutaneously into C57/black mice (N=5). At five days after virusadministration, four out of five mice died in the group receiving wildtype HSV-1. One mouse from the group receiving wild type HSV-2 died.However, none of the mice died in the group injected with FusOn-H2.These results indicate that although extremely potent at killing tumorcells, FusOn-H2 was significantly less toxic than the parental wild typeHSVs to the receiving hosts, and in specific embodiments was safe forclinical application.

Example 8 Ability of FusOn-H2 to Induce Apoptosis

The present example shows that the modified HSV-2 virus (FusOn-H2), asdescribed in the present invention, can efficiently induce apoptosis ininfected and by-stander tumor cells, providing an additional tumordestroying mechanism.

African green monkey kidney (Vero) cells, SW403 and SW480 cells (humancolon cancer cell lines), and A549 cells (a human lung carcinoma cellline) were obtained from the American Type Culture Collection(Rockville, Md.). EC9706, a human esophageal cancer cell line wasprovided by Dr. Mingrong Wang (Chinese Academy of Medical Sciences).SKOV3 cells, a human ovarian cancer cell line, was provided by Dr.Robert Bast (the M. D. Anderson Cancer Center). U20S cells, a humanosteosarcoma line, was provided by Dr. Lawrence Donehower. All of thecells were cultured in DMEM containing 10% fetal bovine serum (FBS).

FusOn-H2 was derived from the wild-type HSV-2 strain 186 (wt186) and itsconstruction is described in Example 1. The construction of Baco-1, anHSV-1-based oncolytic virus is described in U.S. patent application Ser.No. 10/397,635. Viral stocks were prepared by infecting Vero cells with0.01 plaque-forming units (pfu) per cell. Viruses were harvested 2 dayslater and purified as described (Nakamori et al., (2003) Clinical CancerRes. 9(7): 2727-2733). The purified viruses were titrated, aliquoted andstored at −80° C. until use.

Viral Growth Characterization

Cells were seeded in triplicate into 24-well plates at 50% density. Nextday, cells were infected with the viruses at 1 pfu/cell for 1 h. Cellswere washed once with PBS to remove unabsorbed and uninternalizedviruses before fresh medium was added. Cells were harvested at 24 hafter infection. Viruses were released by repeated freezing and thawingand sonication. Virus titers were determined on Vero cells by a plaqueassay.

Hochest Dye Staining of Infected Cells and Quantification of ChromatinCondensation

Cells seeded in 24 well plates were infected next day with FusOn-H2,wt186 or Baco-1 at 10 pfu/cell or mock-infected. Twenty-four h afterinfection, the cells were stained with Hochest dye 33358 (Sigma-Aldrich,Mo.) at a final concentration of 1 μg/ml for 30 min at 37° C. beforephotomicrographs were taken under a fluorescent microscope.

DNA Laddering Assay

Cells were seeded into 6-well plates at 70% density. Next day, cellswere infected with virus at 10 pfu/cell. Twenty-four h after virusinfection, cells were harvested and DNA was extracted from the cellswith DNAzol reagent (Invitrogen, Calif.). The extracted DNA was treatedwith RNase (100 μg/ml) before subjecting to phenol:chloroform extractionand ethanol precipitation. DNA was then loaded to 1% agarose gels forelectrophoreses and visualization under UV illumination after stainingwith ethidium bromide.

Expression of EGFP Corresponds to Chromatin Condensation

Cells seeded in 12 well plates were infected next day with FusOn-H2 at 1pfu/cell. Hochest dye staining for chromatin condensation was done asdescribed above. The overlay of micrographs from the same field withdifferent fluorescent lights were done by using Spot Image Software(Diagnostic Instrument, Inc, Ill.). The GFP positive and GFP negativeapoptotic cells were separately counted in the same fields. About 100apoptotic cells were counted in each field. A total of 3 fields werecalculated for proving the by-stander effect induced by FusOn-H2infected cells.

Terminal Deoxynucleotidyltransferase-Mediated Nick End Labeling (Tunel)Assay.

Female Hsd athymic (nu/nu) mice (obtained from Harlan, Indianapolis,Ind.) were kept under specific pathogen-free conditions and used inexperiments when they attained the age of 5 to 6 weeks. EC9706 cellswere harvested from subconfluent cultures by a brief exposure to 0.25%trypsin and 0.05% EDTA. After trypsinization was stopped with mediumcontaining 10% FBS, the cells were washed once in serum-free medium andresuspended in PBS. On day 0, 5×10⁶ EC9706 cells were inoculated intothe right flank of nude mice. Two weeks after tumor cell implantation,when the tumors reached approximately 5 mm in diameter, mice received asingle intra-tumor injection of 3×10⁶ pfu of FusOn-H2 or Baco-1 in avolume of 100 μl, or the same volume of PBS. The tumors were measuredweekly and their volumes determined by the formula: tumor volume[mm³]=(length [mm])×(width [mm])²×0.52. For Tunel assay, mice wereeuthanized by CO₂ exposure 3 days after intra-tumor injection of 1×10⁷pfu of FusOn-H2 or Baco-1 viruses. Tumor tissues were explanted andsectioned for Tunel staining.

FusOn-H2 Induces Apoptosis in Human Tumor Cells of Different TissueOrigins

Due to the anti-apoptotic activity of certain HSV-2 gene products,infection with HSV-2 does not routinely induce apoptosis unless viralprotein synthesis is blocked with translation inhibitors such ascycloheximide (Aubert et al., (1999) J Virol 73(12): 10359-70). The PKdomain of the ICP10 gene from HSV-2 has been identified as one of theviral gene products that have anti-apoptotic function, and its deletionfrom the viral genome has been described to render the virus with theability to induce apoptotic death of certain type of somatic cells(Perkins, et al., (2002) J Virol 76(3): 1435-49).

To determine if FusOn-H2 induces apoptotic death of tumor cells, weinfected a panel of human tumor cells of different tissue origins withthe virus at an m.o.i. of 10. An oncolytic virus derived from HSV-1,Baco-1, was included as a control. Among the tumor cells, EC9706 is ahuman esophagus cancer cell line, SKOV3 is a human ovarian cancer cellline and SW403 and SW480 are human colon cancer cell lines. The cellswere seeded in 6-well plates and infected with the viruses the next day.Twenty-four h after infection, the cells were stained with Hochest dye33358. Infection of tumor cells with FusOn-H2 induced extensivechromatin condensation, indicative of apoptosis. This was evident by theappearance of intense and compact blue nuclear staining in FusOn-H2infected cells. Overall, over 80% of tumor cells infected with FusOn-H2showed chromatin condensation. Uninfected tumor cells showed very littleor no such apoptotic features. Infection of these tumor cells witheither the parental wild type HSV-2 (wt186) or Baco-1 did notsignificantly increase the background level of blue fluorescent stainingfor the chromatin condensation.

To further validate the capability of FusOn-H2 to induce apoptosis intumor cells, DNA fragmentation was analyzed. Three tumor cells that wereused in the previous experiment were infected with viruses at 10pfu/cell or mock-infected. At 24 h post-infection, cells were harvested.DNAs were extracted from the cells and separated in a 1% agarose gel.There was obvious laddering in the wells where FusOn-H2 infectedmaterials were loaded. This laddering was not detected in the wellswhere DNA sample from either wt186 or Baco-1-infected cells were loaded,thus confirming the result of chromatin condensation presented above.Together, these results demonstrate that infection of FusOn-H2efficiently induces apoptosis in these human tumor cells, while neitherthe parental wild type HSV-2 nor an HSV-1-based oncolytic virus has sucha property.

Infection of FusOn-H2 Also Induces Apoptotic Death of by-Stander Cells

As FusOn-H2 carries the gene encoding the enhanced green fluorescentprotein its infectivity could be easily determined under a fluorescentmicroscope. During the inter-exchange of fluorescent detection ofchromatin condensation and infectivity, we noticed an obviousdiscrepancy between the percentage of cells showing the blue fluorescentchromatin condensation and the cells showing GFP staining. When theabsolute number of tumor cells showing chromatin condensation and GFPexpression was enumerated, the ratio was approximately 2.6:1. Thisresult indicates that there was a substantial by-stander apoptoticeffect on the surrounding tumor cells of FusOn-H2 infection.

FusOn-H2-Induced Apoptosis Accelerates Tumor Cell Death and CompromisesVirus Replication within the Tumor Cells

An obvious difference was also noted with regard to the time when cellsshowed the cytopathic effect (CPE) between the tumor cells infected withFusOn-H2 and the oncolytic virus derived HSV-1. Tumor cells infectedwith FusOn-H2 at a dose of 1 pfu/cell usually showed full CPE within 24h, while the tumor cells infected with Baco-1 at the same dose lookedlargely normal morphologically. They usually did not show obvious signof CPE until more than 72 h after infection. The typical CPE, includingcell round up and detachment from each other, could be readily seen inthe wells infected with FusOn-H2 at 24 h after infection, while thecells infected with Baco-1 looked essentially like the mock-infectedcells even at 48 h after infection. These results indicate that theapoptotic cell death induced by FusOn-H2 occurred immediately followingvirus infection, while it took a significantly longer time for theoncolytic effect of virus replication to occur.

Apoptotic Tumor Cell Death is an Important Anti-Tumor Mechanism of theVirus In Vivo

The anti-tumor activity of FusOn-H2 in vivo against tumor xenograftsestablished from one of the tumor cells used in the previous experimentswas evaluated. Baco-1 was included in this experiment so that thetherapeutic effect of these two viruses could be directly compared.Tumor xenografts were established on the right flank though subcutaneousinjection of 5×10⁶ freshly harvested EC9706 cells. When the tumor sizereached approximately 5 mm in diameter, mice received a singleintra-tumor injection of either viruses (FusOn-H2 or Baco-1) at a doseof 3×10⁶ pfu, or PBS as a control. The tumors were measured regularlyfor 6 weeks and the tumor growth ratio was determined by dividing thetumor volume before therapy with those obtained at different time pointsafter therapy. Therapeutic administration of FusOn-H2 essentiallystopped the tumor growth within one week. Afterwards the tumor startedto shrink and by the end of the experiment, the average tumor size wasonly about the half of the size before viro-therapy and over half of themice were completely tumor-free. When compared with the PBS control,administration of Baco-1 did not show any therapeutic effect until week3. However, it seemed the tumor shrinkage was only transient, as thetumor started to grow again at day 35. Overall, the therapeutic effectof FusOn-H2 was significantly stronger than that of Baco-1 at all of thetime points evaluated (p<0.05), despite the fact that it has limitedreplication capability in this tumor cell due induction of apoptosis.These results indicated that the apoptotic death and accompanyingby-stander effect induced by FusOn-H2 was likely a major anti-tumormechanism in this in vivo study.

Example 9 Tumor Destruction by FusOn-H2 Induces Potent AntitumorImmunity

The antitumor activity of FusOn-H2 was evaluated in two syngenic tumormodels: murine mammary tumor (4T1 cells) and murine neuroblastoma(Neuro2A cells). In both cases, FusOn-H2 produced a statisticallysignificant antitumor effect that was accompanied by robusttumor-specific immune responses. Presented below are typical data fromstudies in the mammary tumor model.

For this evaluation, 4T1 cells were utilized, which are non-immunogenic,highly malignant and highly metastatic in syngenic BALB/c mice (Aslaksonand Miller (1992) Cancer Res 52(6): 1399-405; Pulaski andOstrand-Rosenberg (1998) Cancer Res. 58(7): 1486-93). 4T1 cells (105)were orthotopically injected into the mammary fat pad of immunecompetent BALB/c mice to establish the orthotopic tumor. Mice were leftfor 10 days, after which lung metastases were detectable in more than90% of the group. Tumor-bearing mice were then divided into 3 groups(n=10 each) and injected intratumorally with 1×10⁷ pfu of eitherFusOn-H2, or other oncolytic HSVs derived from HSV-1, including thedoubly fusogenic Synco-2D that was previously shown to induce effectiveantitumor immunity in this model (Nakamori, Fu et al., (2004) Mol. Ther.9(5): 658-665). Tumor masses at the orthotopic site were measured weeklyfor 2 weeks, after which the mice were killed for immunological assaysand for evaluation of lung metastases (enumerated under a dissectingmicroscope after Indian ink infusion). For immunological assays, thesplenocytes were prepared from the explanted spleens and stimulated withirradiated 4T1 cells in vitro for 5 days before being used for thefollowing assays: 1) tumor-specific CTL activity (with either 4T1 cellsor a syngenic sarcoma cell line Meth-A as target cells) by the 51Crrelease assay; 2) Elispot analysis of mouse IFN-γ-secreting cells, usinga detection kit purchased from BD Biosciences; 3) quantification ofcytokine secretion (for both Interferon-γ and IL-10). The results showedthat local intratumor administration of FusOn-H2 produced asignificantly better therapeutic effect than did other viruses, not justagainst the orthotopic tumor, but also against distant lung metastases.As compared with Baco-1, Synco-2D was able to inhibit the growth of theorthotopic and metastatic tumors, a result similar to our previousobservation (Nakamori, Fu et al., (2004) Mol. Ther. 9(5): 658-665).However, FusOn-H2 is apparently even more effective than Synco-2D intreating this tumor. The accompanying antitumor immune responses inducedby FusOn-H2, including the tumor-specific CTL activity and frequency andcytokine release, were also more prominent than that of Synco-2D,indicating their contribution to the elimination of local and metastatictumors.

Example 10 Plaque Forming Assay for Determining Viral Titer

After viral stocks were prepared, the viral titer was determined using aplaque forming assay as previously described (see, Lancz G J. (1974).Arch Virol., 46, 36-43). Vero cells are trypsinized, counted, and platedinto six well plates at 4×10⁵ cells per well and incubated at 37° C.with 5% CO₂ and 90% humidity and cultured for 24 hours. Next day, thevirus is serially diluted 1:10 in 1× Minimal Essential Medium (MEM) togive six concentrations of 10⁻³ to 10⁻⁸. The media is then aspiratedfrom the wells and 0.5 ml of virus dilution is added to each well intriplicate. The plates are then incubated for 1 h with shaking everyfifteen min. After the incubation period, the virus solutions areaspirated and 2 mls of MEM containing 1% agarose is added to each welland the plates are incubated for three days, after which the cells arestained with a solution containing 0.1% crystal violet and 20% ethanol.At the end of the 30 minute incubation period, the stain is aspirated,and plaques counted using a stereomicroscope at 10× magnification. Viraltiter is then expressed as plaque forming units per ml.

1. Use of a compound, comprising a Herpes Simplex Virus Type 2 (HSV-2)having a modified ICP10 polynucleotide therein, said modified ICP10polynucleotide encoding an ICP10 polypeptide having ribonucleotidereductase activity and lacking protein kinase activity, in themanufacture of a medicament for selectively killing cancer cells in ahost.
 2. The use according to claim 1, wherein the ICP10 polynucleotideis modified by deleting a portion of the protein kinase domain of theICP10 polynucleotide.
 3. The use according to claim 1, wherein the ICP10polynucleotide is modified by the insertion of a polynucleotide encodinga reporter protein selected from the group consisting of greenfluorescent protein, β-galactosidase, luciferase, and Herpes SimplexVirus thymidine kinase (HSV tk).
 4. The use according to claim 1,wherein the ICP10 polynucleotide is modified by the insertion of apolynucleotide encoding an immunomodulatory gene selected from the groupconsisting of tumor necrosis factor, interferon alpha, interferon beta,interferon gamma, interleukin-2, interleukin 12, GM-CSF, F42K, MIP-1,MIP-1β, and MCP-1.
 5. The use according to any of the claims 1 to 4,wherein the modified ICP10 polynucleotide is operably linked to aconstitutive promoter.
 6. The use according to any of the claims 1 to 5,wherein the ICP10 polynucleotide is further modified by replacing atleast part of the endogenous protein kinase activity domain with apolynucleotide encoding a fusogenic membrane glycoprotein.
 7. The useaccording to claim 6, wherein the fusogenic membrane glycoprotein isselected from the group consisting of: a gibbon ape leukemia virusenvelope fusogenic membrane glycoprotein; a murine leukemia virusenvelope protein; a retroviral envelope protein lacking the cytoplasmicdomain; a measles virus fusion protein; an HIV gp160 protein; an SIVgp160; a retroviral Env protein; an Ebola virus Gp; and, an influenzavirus haemagglutinin.
 8. The use according to claim 1, wherein themedicament selectively kills cancer cells by fusion of the cancer cellmembranes (syncytial formation).
 9. The use according to claim 1,wherein the medicament selectively kills cancer cells by inducingapoptosis in infected and by-stander tumor cells.
 10. The use accordingto claim 1, wherein the medicament is administered intratumorally. 11.The use according to claim 1, wherein the medicament is administeredsystemically.
 12. The use according to claim 1, wherein the medicamentis administered intraperitoneally.
 13. A method of generating an HSV-2virus having oncolytic and fusogenic properties, comprising the stepsof: modifying the ICP10 polynucleotide, wherein the modified ICP10polynucleotide encodes for an ICP10 polypeptide having ribonucleotidereductase activity and lacking protein kinase activity, and operablylinking the modified ICP10 polynucleotide with a constitutively activepromoter.
 14. A composition comprising a Herpes Simplex Virus Type 2(HSV-2) having oncolytic and fusogenic properties wherein the HSV-2comprises a modified ICP10 polynucleotide, said modified ICP10polynucleotide encoding an ICP10 polypeptide having ribonucleotidereductase activity and lacking protein kinase activity, and wherein themodified ICP10 polynucleotide is operably linked to a constitutivepromoter.
 15. The composition of claim 14, wherein the HSV-2 furthercomprises a polynucleotide encoding a fusogenic membrane glycoproteinwhich is inserted into the protein kinase activity domain of the ICP10gene, and wherein the fusogenic membrane glycoprotein is selected fromthe group consisting of: a gibbon ape leukemia virus envelope fusogenicmembrane glycoprotein; a C-terminally truncated form of the gibbon apeleukemia virus envelope glycoprotein (GALV.fus); a murine leukemia virusenvelope protein; a retroviral envelope protein lacking the cytoplasmicdomain; a measles virus fusion protein; an HIV gp160 protein; an SIVgp160; a retroviral Env protein; an Ebola virus Gp; and, an influenzavirus haemagglutinin.
 16. The composition of claim 14, wherein the HSV-2comprises a polynucleotide inserted into the protein kinase activitydomain of the ICP10 gene, the polynucleotide encoding a reporter proteinselected from the group consisting of green fluorescent protein,β-galactosidase, luciferase, and Herpes Simplex Virus thymidine kinase(HSV tk).
 17. The composition of claim 14, wherein the HSV-2 comprises apolynucleotide inserted into the protein kinase activity domain of theICP10 gene, the polynucleotide encoding an immunomodulatory geneselected from the group consisting of tumor necrosis factor, interferonalpha, interferon beta, interferon gamma, interleukin-2, interleukin 12,GM-CSF, F42K, MIP-1, MIP-1β, and MCP-1.
 18. The composition of claim 14,wherein the HSV-2 further comprises a therapeutic polynucleotideinserted into the protein kinase activity domain of the ICP10polynucleotide, wherein the therapeutic polynucleotide is selected fromthe group consisting of HSV tk, cytosine deaminase, and caspase-3. 19.The composition of claim 14, wherein the constitutively active promoteris an immediate early cytomegalovirus promoter.
 20. A compositioncomprising an HSV-2 viral vector comprising a modified ICP10polynucleotide that encodes for a polypeptide having ribonucleotidereductase activity and lacking protein kinase activity, and wherein themodified polynucleotide is operably linked to a constitutive promoter